Quantification of Surface Ligands on NaYF4 Nanoparticles by Three

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Quantification of Surface Ligands on NaYF4 Nanoparticles by Three Independent Analytical Techniques Lemuel Tong, Elsa Lu, Jothirmayanantham Pichaandi, Pengpeng Cao, Mark Nitz, and Mitchell A. Winnik* Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada S Supporting Information *

ABSTRACT: There have been important advances in characterizing the surface coverage of ligands on colloidal inorganic nanoparticles (NPs), but our knowledge of ligand coverage on lanthanide NPs is much more limited. The assynthesized NPs are often coated with hydrophobic ligands that need to be replaced with hydrophilic ligands such as poly(ethylene glycol) (PEG) for biomedical applications. The two challenges in terms of characterizing ligand coverage on NPs are first to show that different analytical methods give consistent results and second to show how the sample preparation protocol affects ligand density. Here, we report a quantitative study of the native oleate content of as-synthesized NaYF4 and NaTbF4 NPs, as well as the surface coverage after ligand exchange with three methoxyPEG-monophosphates with Mn = 750, 2000, and 5000 Da. For NaYF4, we obtained consistent results for both oleates and PEGs by three independent methods (TGA, 1H NMR, and ICP-AES). The oleate coverage was very sensitive to the sample isolation/purification protocol, with a high surface coverage (5.5 to 8 nm−2) for ethanol/hexane sedimentation/ redispersion but only 2 nm−2 if THF was used in place of hexanes. The surface coverages PEG750 (∼1.1 nm−2), PEG2000 (∼1.7 nm−2), and PEG5000 (∼0.2 nm−2) suggest that corona repulsion limits the number of PEG5000 molecules that can graft to the surface. For NaTbF4 NPs, we compared the surface coverage of PEG2000-monophosphate with a PEG2000-tetraphosphonate ligand shown to provide enhanced colloidal stability in PBS buffer. We found the surprising result that the footprints of these ligands were comparable, suggesting that there was insufficient room for all four phosphonate groups of the tetradentate ligand to bind simultaneously to the NP surface.

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INTRODUCTION Over the past 20 years there has been an explosive growth in interest in colloidal inorganic nanoparticles (NPs).1 Examples include metal NPs (especially gold and silver),2 metal oxide NPs (particularly iron oxide),3 quantum dots (QDs),4 and lanthanide NPs such as lanthanide doped NaYF4 NPs. In the biomedical field, gold NPs have been employed as Raman imaging agents,5 superparamagnetic iron oxide NPs (SPIONs), and NaGdF4 NPs as contrast agents for magnetic resonance imaging (MRI),6 and quantum dots as fluorescent markers.7 The physical and chemical properties of these NPs have been exploited by varying their size and shape as well as by modifying their surfaces with functional ligands and protein-repellent polymers such as polyethylene glycol (PEG). These types of NPs have also been incorporated into micelles and other nanomaterials to create multifunctional reagents for tumor imaging and cancer treatment.8,9 We are interested in developing NaLnF4 (Ln = lanthanide) NPs as reagents for high throughput analysis of biomarker expression in individual cells by mass cytometry (CyTOF). In CyTOF, cells are injected individually but stochastically into the plasma torch of an inductively coupled plasma mass spectrometer equipped with time-of-flight detection.10−12 In the analysis protocol, cells are treated with a cocktail of different antibodies (Abs) in which each type of Ab is labeled with a different metal isotope. The abundance of each isotope © XXXX American Chemical Society

determined by CyTOF reflects the number of each targeted biomarker on each cell. Current reagents for mass cytometry are metal chelating polymers that label each Ab with ca. 200 copies of an isotope. Ln NPs represent an attractive option for increasing the sensitivity of the measurement by increasing the number of Ln ions attached to an Ab.13 The most serious challenge is to modify the surface of these NPs to impart colloidal stability in physiological buffers and biological media and also to suppress or minimize nonspecific interaction with cells lacking the targeted biomarker. There have been several reports on the surface modification of lanthanide based NPs. However, we lack a detailed understanding of ligand binding to the surface of these NPs and how this affects the colloidal stability of the NPs in buffers and biological media. Here, we examine and compare different analytical techniques for quantifying the coverage of organic and polymeric ligands on the surface of these NPs. To put our work in context, we first provide a brief review of other NPs, particularly gold, CdSe, and iron oxide NPs, for which there has been intense recent activity devoted to understanding the details of ligand interaction with the NP surface. One important issue is ligand stoichiometry. The implication of Received: June 10, 2015 Revised: June 12, 2015

A

DOI: 10.1021/acs.chemmater.5b02190 Chem. Mater. XXXX, XXX, XXX−XXX

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Chemistry of Materials stoichiometry is that there are specific sites of ligand-headgroup attachment, and that the surface density of ligands (ligands/nm2) is determined by a balance of the spacing of attachment sites on the surface and the steric requirements of the ligand tail. Other factors come into play. For example, while a high degree of ligand exchange can often be achieved, ligand replacement is normally not quantitative. Different sample preparation protocols can lead to samples with different ligand surface densities.14 It is well documented that the strength of the NP−ligand bond can significantly be increased by using multivalent ligands with a larger number of coordinating groups. It is more difficult to characterize the footprint of a multidentate ligand on the surface than to characterize the area occupied by a monodentate ligand. There is also concern about the methods used to determine the ligand coverage and the displacement of one ligand by another. It would be very useful if one could show for any given NP sample that different analytical methods give consistent values of the number of ligands per NP. For gold NPs, functional ligands such as 11-mercaptoundecanoic acid and thiol-modified PEG (PEG-SH) ligands can be introduced by replacing weakly physisorbed citrate ions by the stronger binding thiolate groups. Several recent papers describe methods to quantify the coverage density of these ligands. For example, Rahme et al. used thermal gravimetric analysis (TGA) to quantify the surface ligand coverage of PEG-SH of various molecular weights on different sizes of gold NPs.15 Hinterwirth et al. describe an inductively coupled plasma mass spectrometry (ICP-MS) approach to measure the S/Au ratio for gold NPs of different sizes.16 Xia et al. examined gold NPs carrying functional PEGs of the form HS-(PEGn)-NH2, where n refers to the mean degree of polymerization of the EG repeat unit.17 Rather than using TGA or 1H NMR to determine the PEG content of their sample, they compared three different methods for determining the number of −NH2 groups in the sample. A striking feature of their experiments is that the quantitative results obtained from each of the three methods were significantly different. Graf and co-workers compared gold NPs prepared in the absence of chloride ions (“naked” gold NPs) with more traditional gold NPs prepared using HAuCl4 as the gold source.18 They showed that in the latter case, chloride ions remain bound to the surface of the NPs after ligand exchange with thiols. Following displacement with PEG-SH or multidentate PEG-dithiol or PEG-trithiol ligands, the residual chloride ions on the surface enhance the degradation rate of the NPs when exposed to cyanide ion. Much of the recent attention to ligand binding to CdSe quantum dots has involved NPs with carboxylate (e.g., oleate) groups at the surface. 1H NMR has proved to be a powerful tool to quantify the number of ligands and to characterize ligand exchange pathways. The as-synthesized QDs have an excess of Cd ions at the surface. The Hens group has shown that the unsatisfied valence sites of the Cd ions bind carboxylate ions and that these carboxylates do not spontaneously dissociate.19,20 Ligand replacement with carboxylic acids occurs via a process involving preassociation of the carboxylic acid with the QD followed by proton transfer to the departing carboxylate ligand. Owen and co-workers examined exchange with soft ligands such as amines and phosphines.21 They showed that the displaced ligands departed as Cd salts (i.e., Cd(oleate)2) and, in the process, reduced the number excess of Cd ions at the QD surface. They go on to comment that the QDs can have a wide range of surface metal coverage and that the stoichiometry obtained for a QD sample is sensitive to the reagents and concentrations used to separate the nanocrystals from unreacted M(O2 CR) 2

precursors. These stoichiometries (and the associated ligand coverage) are not fixed for a given NP size and depend upon the isolation method. They emphasize, as will become important in the results we present below, that these aspects of NP composition are sensitive to the solvents and concentrations as well as to the number of precipitation cycles used in sample purification. Catechols, carboxylates, and phosphonates are typical ligands for iron oxide NPs. For some ligands, we have a good understanding of how the ligand binds to the NP surface. Daou et al. used a combination of FTIR, X-ray photoelectron spectroscopy (XPS), and Mossbauer spectroscopy to look at the binding of phosphate anions to 40 nm magnetite NPs.22 They show that at low pH, two oxygen atoms of the phosphate bind to adjacent Fe atoms on the NP surface but that at lower pH only one oxygen atom is bound. Polito et al. developed a magic angle spinning method to obtain fine-structure resolved 1H NMR spectra of paramagnetic iron oxide NPs.23 For 7 nm magnetite NPs, they could distinguish tightly bound ligands from excess free ligands in solution and show how more complex ligands were bound to the particle surface. In the context of our interest in NaLnF4 NPs, we were particularly interested in a recent paper by Mefford and coworkers, who report a radiotracer (14C) method for quantifying oleates on the surface of 10 nm diameter Fe3O4 NPs and for studying their replacement by PEG chains terminated with amino, carboxylate, phosphonate, or catechol groups.24 One of their goals was to overcome a shortcoming of TGA measurements, which determine mass loss from NPs without indicating which ligands were present. The trace of radiolabeled oleate allows them to identify oleate associated with the NPs and allows them to monitor residual oleates bound to the NPs after ligand exchange with functional PEGs. The as-prepared NPs, after purification by dialysis in chloroform, had an oleate content (bound plus excess oleate) corresponding to 20 molecules/nm2. This high value was reduced to 12 oleates/nm2 by passing the NPs through a gel permeation chromatography column packed in toluene. Ligand exchange with PEG-dopamine and PEGnitrodopamine displaced almost all of the oleates from the NP surface. In contrast, after a similar attempt to exchange the surface ligands with PEG-COOH, the authors conclude that all of the organic material on the particles is oleate and not PEG. NaLnF4 NPs are more complicated than the materials referred to above. Metal NPs have a single component. QDs and metal oxides are binary systems. As mentioned, for some of these systems (e.g., PbS, PbSe, CdS, and CdSe), there is evidence that the metal ions are enriched at the NP surface. NaLnF4 NPs are ternary systems, and doped NaLnF4 NPs (e.g., NaYF4:Yb,Er) have even more components. The distribution of Na and Ln ions in the surface of NaLnF4 NPs remains a mystery. These NPs are normally synthesized at high temperature (ca. 300 °C) in a mixture of oleic acid (OA) and octadecene (ODE).8 Some authors use a mixture of oleic acid and oleylamine (OM). The NPs can be formed in a cubic (α) crystalline phase or a hexagonal (β) crystalline phase. For many applications, the β phase is preferred. These NPs form stable colloidal solutions in hydrocarbon media due to the presence of the hydrophobic ligands bound to their surface. One would like to know how ligands such as OA are bound to the NP surface. One would like a deeper understanding of how these oleate-capped NPs undergo ligand exchange. From an analytical chemistry perspective, one would like to assess the reliability of different methods for determining the ligand content of a NP sample, and from a B


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Chemistry of Materials

analyzer. NPs were heated to 100 °C in air at a heating rate of 10 °C/min and held isothermally for 30 min to remove traces of organics and moisture followed by heating at 10 °C/min to 800 °C. The mass fraction of ligands was calculated by the percent mass loss of the ligands. As a control, the pure ligands were subjected to the same TGA protocol. Incomplete decomposition of the pure ligands was taken into account (eq S1, Supporting Information). 1 H NMR Analysis. NPs for NMR analysis were prepared as follows: oleate-capped NPs were purified by three redispersion−sedimentation cycles with cyclohexane and ethanol by centrifugation (5000 rpm, 1398g, 5 min). In the last washing step, the NPs were sedimented with d6-ethanol. The supernatant was discarded, and the NPs were briefly airdried before redispersion in d8-toluene (300 μL). PEGylated NPs were purified by spin filtration via centrifugation (3000 rpm, 805g, 30 min). The purified NPs were freeze-dried overnight to remove residual water before redispersion in D2O (300 μL). Appropriate amounts of internal standard were added into sample. NMR spectra of the pure ligands and the internal standards are presented in Figures S2 and S3 (Supporting Information). All NMR spectra were recorded with a Varian DD2 700 MHz NMR spectrometer. Samples were measured at 20.0 ± 0.5 °C in 0.3 mm tubes. A standard inversion recovery method was used to measure the proton T1 relaxation times. The native ligands and the internal standards were measured individually in their respective solvents to determine the T1 relaxation time prior to measurement with the ligand capped NPs. These values are collected in Table S1 (Supporting Information). Standard 1D proton NMRs were acquired with at least 5 × T1 of the component with the highest T1 relaxation time (number of scans = 16, number of runs = 3, pulse angle = 45°, observe pulse = 2.45 μs, and acquisition time = 2.936 s). Each sample was measured in triplicate. The NMR spectra were processed with MestReNova 8.1.4 software. The ligand weight fraction (wl) was calculated with eq S2 (Supporting Information). Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). NPs for ICP-MS analysis were prepared as follows: oleate and PEGylated NPs were dried in a vacuum oven and freeze-dried, respectively, to remove residual organic solvents and water. Dried NPs (∼3 mg) were digested in aqua regia (1 mL, HCl/HNO3 (1:3 v/v), Seastar Chemicals Inc.) overnight in sealed polypropylene tubes (2 mL). Aliquots (150 μL) of the digestion solution were diluted with deionized water (Millipore EMD) to adjust the concentration of HNO3 to 2 vol % from aqua regia in three separate propylene tubes (2 mL). Subsequent 10-fold dilutions were made with 2% HNO3 until ppb concentrations were obtained. The dilutions were repeated in triplicate. All samples were measured with an ELAN 9000 ICP-MS for 89Y analysis. The standard solution was prepared by a series of 10-fold dilutions of yttrium standard solution (1000 mg/L, 2% HNO3, PerkinElmer) in triplicate to obtain three standard solutions. The blank solution (2 vol % HNO3) was also measured in triplicate. A linear calibration curve was obtained, relating the counts and yttrium concentration using Origin Pro 9. The ligand weight fraction (wl) was calculated with eq S3 (Supporting Information). Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). NP samples were digested with HNO3 overnight and diluted to ppm concentrations and measured in triplicate using an Optima 73100 DV ICP-AES (PerkinElmer). The operating conditions are listed in Table S3 (Supporting Information). The ICP-AES was operated in the axial mode. In the axial mode, the detection limits for Y and P are 0.006 and 0.05 ppm, respectively. The concentrations of Y and P were determined by the average integration under the peak area corresponding to wavelengths Y = 371.029, 324.227, and 360.073 nm and P = 213.617 and 178.221 nm. The phosphorus standard and yttrium standard were prepared by 10-fold dilutions with 2% HNO3 from the Pure Single-Element Standard (1000 μg/mL) (PerkinElmer, Woodbridge, ON). Three sequential standards were used to generate the calibration curve. A sample blank with the same concentration was used as a background as well as a calibration blank. The Y and P concentrations were determined by correlating the peak area with the calibration standards. The ligand density (ρl) was determined by using eq S4 (Supporting Information).

synthetic perspective, one would like to assess how sensitive NP samples are to different isolation and purification protocols. We address some of these issues in the experiments described below. In this article, we quantify the surface density of the native oleate and grafted PEG (MW ∼ 750, 2000 and 5000) ligands on uniform NaYF4 NPs with multiple analytical tools: TGA, 1H NMR, ICP-MS, and ICP atomic emission spectroscopy (ICPAES). We also compare the ligand density between a monodentate and multidentate ligand on NaTbF4 NPs. Our most important conclusions are that different analytical techniques give consistent results but that measured ligand densities on individual samples are sensitive to the sample isolation and purification protocol.

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EXPERIMENTAL SECTION

Materials. NaTbF4 NPs (dTEM = 10 nm) were synthesized as reported previously25 and NaYF4 NPs were synthesized based on a procedure reported by the van Veggel laboratory26 using a setup as shown in Figure S1 (Supporting Information). Details are provided in Supporting Information. MethoxyPEG750 and methoxyPEG2000 monophosphates (mPEG750-OPO3H2 and mPEG2000-OPO3H2)27 and the methoxyPEG-tetraphosphonate25 are the same samples described previously. The synthesis of mPEG5000-OPO3H2 is described in Supporting Information. Purification of Lanthanide Nanoparticles. The NPs were purified from excess ligands by several sedimentation−redispersion cycles using ethanol as a precipitant. An aliquot (4 mL) of the reaction mixture was sedimented with excess ethanol (11 mL) by centrifugation (5000 rpm, 1398g, 5 min). The supernatant was removed, and the sedimented NPs were redispersed with hexanes (0.8 mL). Once again, the NPs were sedimented with excess ethanol (11 mL) and centrifuged (5000 rpm, 1398g, 5 min). The sedimentation−redispersion cycle was repeated once more before the NPs were redispersed in organic solvents such as CHCl3 or hexanes. Ligand Exchange with PEG Ligands. The ligand exchange of methoxyPEG-monophosphate (mPEG-OPO3H2) with NaYF4 NPs was modified from a previous published report.25 In brief, PEG phosphate ligands were dissolved in CHCl3 (30 mg/mL, 3 mL), and the NP dispersion in CHCl3 was adjusted to 20 mg/mL. The NP solution (2 mL) was added dropwise into the PEG mixture and stirred overnight. Displaced OA was removed by 3 sedimentation−redispersion cycles using excess n-hexanes as a precipitant followed by centrifugation (4000 rpm, 1431g, 15 min). The supernatant was discarded, and the sedimented NPs were redispersed in CHCl3 (3 mL). The NPs were then sedimented with excess n-hexanes. After OA was removed, the sedimented NPs were dried in a stream of Ar and redispersed with DI H2O (15 mL). Excess PEG ligands were filtered away with Amicon Ultra spin filters. PEG750 and PEG2000 capped NPs were spin filtered with a 10 kDa molecular weight cutoff (MWCO), and PEG5000 capped NPs were spin filtered with a 30 kDa MWCO. Excess PEG was removed by three cycles of spin filtration via centrifugation (805g, 30 min). After each cycle, the filtrate was removed, and DI H2O was added to the residual solution (15 mL). The purified NPs were then stored at room temperature Nanoparticle Characterization. Characterization of the NaYF4 and NaTbF4 NPs by TEM and XRD is described in Supporting Information. The hydrodynamic diameter of NPs was measured by DLS on a Malvern Zetasizer Nano ZS at 25 °C with a 173° backscatter measurement angle. Samples were filtered through a nylon syringe filter (0.2 μm) prior to measurements, and measurements were conducted in triplicate. OA-capped NPs were dispersed in n-hexanes, and mPEGOPO3H2-capped NPs were dispersed DI water. Z-averaged hydrodynamic diameters (dh) and polydispersities (PDI) were calculated from the first and second cumulants of the autocorrelation decays. Thermogravimetric Analysis (TGA). NP samples were dried in a vacuum oven and lyophilized to remove residual organic solvents and water. Approximately 5 mg of samples was measured in alumina pans and heated in a SDT Q600 (TA Instruments) thermogravimetric C


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Figure 1. TEM images of two identical batches (d ∼18 nm) of NaYF4 NPs (A,E), size histogram (d ∼18 nm) (B,F), and CONTIN plot of the hydrodynamic diameter (dh) by DLS analysis (C,G). TGA profiles of NPs washed with THF (D) and n-hexanes (E). The mass percent of OA on NaYF4 NPs (d ∼18.0 ± 0.8 nm) after washing with hexanes and THF is 25.0 and 8.8 wt %. The oleate densities of NPs washed with hexanes and THF are 8.9 and 2.6 nm−2, respectively.

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RESULTS AND DISCUSSION

We prepared uniform NaYF4 NPs (CV < 5%) with a low aspect ratio (1.10 ± 0.04). The low aspect ratio allows us to approximate their shape as spheres. These NPs were synthesized in a mixture of oleic acid (OA) and 1-octadecene (ODE) using a high temperature coprecipitation method reported by van Veggel and co-workers.28 NPs of different sizes were obtained by varying the reaction time at 300 °C. Powder X-ray diffraction (PXRD) measurements showed that all of the NP samples were in the hexagonal crystal phase. (Figure S4, Supporting Information). We begin by looking at the oleate content of the as-synthesized NaYF4 NPs. In a subsequent section of the article, we examine

In order to examine the ligand content of NaLnF4 NPs in more detail, we turned to undoped NaYF4 NPs that offer some important characteristics for ligand characterization. First, yttrium is one of the few diamagnetic elements in the lanthanide series (besides La and Lu) making these NPs a suitable candidate for 1H NMR analysis. Second, because yttrium contains a single isotope, it is particularly suitable for ICP-MS and ICP-AES analysis. In this way, we can examine the ligand content of these NPs by several independent measurements. D


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Figure 2. Two sizes of NaYF4 NPs synthesized by varying the reaction time at 300 °C. The average sizes of NaYF4 NPs are d ∼16.8 ± 0.6 nm (A,B) and d ∼22.0 ± 0.9 nm (E,F). The hydrodynamic diameter of oleate-capped NaYF4 NPs in hexanes obtained from the intensity weighted DLS is 24.4 ± 0.1 nm (C) and 27.7 ± 0.1 nm (G) washed by three sedimentation−redispersion cycles with hexanes/ethanol. The TGA degradation profile of oleate-capped NPs was performed in air with a mass loss of 24.6% (D) and 14.7% (H).

the ligand content of these NPs after ligand exchange with several different PEG-phosphates and one tetradentate PEG tetraphosphonate. For a given set of spherical NaLnF4 NPs covered with a monolayer of surface ligands, the ligand density (ρl, ligand molecules per nm2) can be determined from the weight fraction of the NP and its ligand components, assuming that the mass of a NP can be calculated from its bulk density (4.21 g/cm3 for NaYF4).29 Hence, the ligand density (ρl, nm−2)

ρl =

wlNAv mNP · M w , l (1 − wl)ANP

(1)

wherein wl is the weight fraction of the ligand, NAv is Avogadro’s number, Mw,l is the molecular weight of the ligand, (1 − wl) is the weight fraction of the NP, ANP is the surface area of a NP, and mNP is the mass of one NP. The weight fraction of ligands was determined by several independent methods as described below. Oleate Content of As-Synthesized NPs. Before any meaningful experiments can be carried out on a newly E


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al. argue that the steric requirements of the alkyl chains in the ligands will prevent coverages from exceeding the density of crystalline alkyl chains (4.9 chains/nm2).31 To obtain an independent assessment of the amount of OA on the NaYF4 NPs, we carried out ICP-MS measurements. A known amount of dried OA-capped NPs were digested and diluted to ppb concentration in triplicate. The yttrium ion (89Y3+) concentration was measured. Using the density and stoichiometry of NaYF4, we calculated the weight fraction of ligands in the sample using eq S4 (Supporting Information). For sample 22E, we found 5.3 ± 1.0 OA nm−2 consistent with the results by TGA. To obtain further information on these OA-coated NPs, we carried out 1H NMR measurements in toluene-d8, comparing the spectrum of oleic acid itself (Figure 3A) with that of sample 17A.

synthesized sample, the NPs have to be separated from the reaction mixture and purified from unbound excess OA. For NaLnF4 NPs, this process involves multiple steps of precipitation with ethanol followed by redispersion in an alkane solvent such as hexanes or a more polar solvent such as tetrahydrofuran (THF). The ethanol precipitant is miscible with the reaction mixture but sediments the NPs. We begin by examining two nearly identical batches of NaYF4 NPs with diameters dTEM = 18.0 ± 0.8 nm (18A) and 17.8 ± 0.7 nm (18E), as measured by transmission electron microscopy (TEM), (Figure 1). In our notation, 18 refers to the particle diameter (dTEM in nm) determined by TEM, and A and E refer to the NPs shown in the TEM images in Figure 1A and E, respectively. These samples were purified separately by three sedimentation−redispersion cycles with either THF (18A) or hexanes (18E). The dynamic light scattering (DLS) results shown in Figure 1C and G emphasize the similarities between the two samples. Both DLS measurements were carried out in hexanes, but for 1C, the sample was rapidly transferred to hexanes after the last precipitation from THF with ethanol. Both samples show hydrodynamic diameters of dh = 26 nm. By comparing the hydrodynamic diameters with the TEM diameters, we infer a thickness of the OA corona of about 4 nm for both samples. Aliquots of these samples were dried in a vacuum oven and then examined by TGA in an air atmosphere. Control experiments (Figure S5, Supporting Information) in which we compared the decomposition of OA under N2 and in air showed residual residual carbonaceous material after heating to 800 °C in nitrogen but clean decomposition at lower temperatures in air. We take the mass loss between 100 and 600 °C to be due to the decomposition of the oleate ligands and note that the mass loss for the THF-washed NPs is substantially smaller (18A, 8.8%) than that of the hexanes-washed sample (18E, 25.0%). It is clear that washing with THF removed more oleate or oleic acid molecules from the sample than a similar treatment with hexanes. The two samples also have a different texture. The dried the sample washed with hexanes was a waxy solid, whereas the sample washed with THF was a fine powder. Converting the mass loss to surface coverage, we learn that the coverage of OA on NaYF4 NPs was reduced from 8.9 to 2.6 OA/nm2 when washed with THF instead of n-hexanes. Consistent with this observation was the reduced colloidal stability of the NPs washed with THF. After three sedimentation−redispersion cycles with THF, the NPs tended to precipitate when allowed to stand in THF. Therefore, to maintain the colloidal stability of the NPs in subsequent experiments, we washed the NaYF4 NPs with hexanes. To follow up on these observations, we prepared two new samples of NaYF4 NPs, characterized by TEM (Figure 2) by dTEM = 16.8 ± 0.6 nm and CV = 3.6% (17A) and dTEM = 22.0 ± 0.9 nm and CV = 4.1% (22E) and purified them by three sedimentation−redispersion cycles with hexanes/ethanol. Dispersions of these NPs in hexanes exhibited hydrodynamic diameters dh = 24.4 ± 0.1 and 26.5 ± 0.1 nm, respectively. The TGA degradation profiles in Figure 2 show that the smaller NPs were characterized by the larger percentage mass loss. Calculation of the OA coverage density led to values of 8.2 ± 0.2 nm−2 for 17A and 5.9 ± 0.2 nm−2 for 22E. These coverage values are high and exceed the theoretical limit for carboxyl group binding on surfaces, where a −COO group is predicted to occupy approximately 0.2 nm2, which corresponds to 5 carboxylates nm−2.30 From another perspective, Anderson et

Figure 3. 1H NMR spectra in toluene-d8 of (A) oleic acid and (B) oleatecapped NaYF4 NPs 17A (dTEM = 17 nm) with the internal standard dimethyl terephthalate. *, represents peaks from toluene-d8. †, the weak sharp peak at 1.4 ppm is due to residual cyclohexane.

In this case, to avoid unwanted peaks in the NMR spectrum, the NP sample was washed with three sedimentation−redispersion cycles using ethanol/cyclohexane in the first two cycles and ethanol-d6 as the precipitant in the third cycle. In this way, we minimize ethanol peaks in the spectrum after redispersion in toluene-d8. The peaks in Figure 3A for OA itself are sharp, which is typical of a mobile small molecule in solution. In contrast, the peaks in the NP sample are broadened, a consequence of restricted movement associated with binding to the NP surface. Peak broadening is so pronounced that there is no detectable signal from the olefinic protons (5.4 ppm in A) of the double bond. Even though this sample was determined by TGA to have an unexpectedly large OA content, there is no evidence of a mobile fraction of OA molecules in the sample. To quantify the OA composition of the NP sample, we ran the NMR in the presence of dimethyl terephthalate (DMT) as an internal standard. We determined T1 relaxation times (Table S1, Supporting Information) to ensure that spectra were accumulated with sufficient delay times to ensure accurate peak integrations. By comparing the integration of the peaks of the internal standard DMT such as the aromatic peaks (aromatic protons at 7.95 ppm; methyl ester protons at 3.47 ppm) with the methyl peak of OA (t, 0.92 ppm), we can calculate the OA content of the sample (Figure S6, Supporting Information). In this way, we find values for the OA ligand density of 7.9 ± 0.1 nm−2 for sample 17A and 5.1 ± 0.5 nm−2 for sample 22E. The values of the OA ligand density determined by each of the different measurement techniques are summarized in Table 1. As mentioned above, the surface coverage determined by these F


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Chemistry of Materials Table 1. Summary of Results of Ligand Density Measurements on NaYF4 NPs 17A and 22Ea NaYF4 17A (dTEM = 17 nm)

NaYF4 22E (dTEM = 22 nm)

ligand density (nm−2) ligand oleic acid

M (g/mol) 282.5

b

TGA 8.2 ± 0.2

ligand density (nm−2) b

NMR 7.9 ± 0.1

b

TGA 5.9 ± 0.2

NMRb 5.1 ± 0.5

ICP-MSc 5.3 ± 1.0

a

The standard error associated with each technique was determined by error propagation using the standard deviations of each measurement carried out in triplicate. bTGA traces and NMR spectra for each sample were measured separately three times. cICP-MS measurements: a digested NP sample was subjected to dilution in triplicate followed by separate measurements.

Figure 4. TEM image of NaYF4 NPs (dTEM = 17 nm) sample 17A after ligand exchange with (A) mPEG750-OPO3H2, (B) mPEG2000-OPO3H2, and (C) mPEG5000-OPO3H2. (D) CONTIN plots from DLS measurements. Values of dh and polydispersities for samples showing that monomodal distributions were evaluated from the first and second cumulants of the autocorrelation decay. For mPEG750-OPO3H2,, dh values were estimated from the peak positions in the CONTIN plot. The dh values calculated in this way for NaYF4 NPs capped with mPEG750, mPEG2000, and mPEG5000 are 37 and 119; 41.6 ± 0.5 and 48.9 ± 0.3 nm, respectively. (E)TGA profile of NaYF4 NPs coated with mPEG750, mPEG2000, and mPEG5000. The mass losses of ligands are summarized in Table 2. The degradation profiles of the pure ligands are given by the black lines, whereas those of the ligand-capped NPs are given by the red lines.

peak. The peak at the larger diameter (dh ≈119 nm) is likely due to aggregation. The coverage density of PEG ligands on sample 17A was first examined by TGA analysis. The degradation profiles of the mPEG-capped NaYF4 NPs as well as the pure ligands are shown in Figure 4. Unlike OA which decomposes completely at high temperature, the mPEG-monophosphates generate a nonvolatile decomposition product whose mass corresponds to the mass fraction of phosphate on mPEG-monophosphate ligands. To obtain the true PEG ligand content of the NPs, the measured mass losses of the ligand-capped NPs were corrected for the residual mass contributions of the ligands. After ligand exchange with mPEG750, 2000, and 5000, the ligand densities on these 17 nm NaYF4 NPs were calculated to be 1.4 ± 0.1, 1.7 ± 0.1, and 0.17 ± 0.01 nm−2, respectively. (Table S2, Supporting Information). A similar experiment with mPEG2000-capped sample 22E (dTEM = 22 nm) gave a value of 2.0 ± 0.1 ligands/ nm2. (Figure S7, Supporting Information). These data are collected in Tables S3 and S4, Supporting Information. As an independent measurement of the ligand content of these NPs, we carried out ICP-MS measurements as described above

different techniques exceeds the theoretical limit for carboxyl group binding on the NP surface. Thus, we conclude that a significant fraction is of the OA is not directly bound to the NP surface but is likely immobilized due to the intermeshing of the long hydrocarbon chains. NaYF4 Nanoparticles after Ligand Exchange with mPEG-OPO3H2. To transfer the hydrophobic NaYF4 NP sample 17A into an aqueous environment, the native OA ligands were ligand exchanged with three different methoxypoly(ethylene glycol) monophosphate (mPEG-OPO3H2) samples of Mn = 750, 2000, and 5000. The NPs dispersed in CHCl3 were treated with a large excess of the mPEG monophosphates and then purified by sedimentation−redispersion. The TEM images in Figure 4 show that the NPs retain their geometry and their core size (dTEM = 17 nm) after ligand exchange. DLS measurements show an increase in the hydrodynamic diameter, yielding values of dh = 42 nm for mPEG2000-capped NPs and dh = 49 nm for mPEG5000-capped NPs. The CONTIN plot for the mPEG750-capped NPs (Figure 4D) shows a bimodal distribution, from which we estimate a value of dh ≈ 37 nm for the smaller G


Article

Chemistry of Materials Table 2. Summary of Results of Ligand Density Measurements on NaYF4 NPs 17A and 23Ea NaYF4 17A (dTEM =17 nm) ligand density (nm−2) ligand mPEG750-OPO3H2 mPEG2000-OPO3H2 mPEG5000-OPO3H2

M (g/mol) 817 2094 5091

b

TGA 1.4 ± 0.1 1.7 ± 0.1 0.17 ± 0.01

b

NMR 0.84 ± 0.06 1.7 ± 0.1 0.27 ± 0.01

c

ICP-AES 1.1 ± 0.2 1.1 ± 0.1 0.18 ± 0.09

NaYF4 23E (dTEM = 23 nm) ligand density (nm−2) TGAb

NMRb

ICP-MSc

2.0 ± 0.1

1.9 ± 0.5

2.3 ± 0.5

a

The standard error associated with each technique was determined by error propagation using the standard deviations of each measurement carried out in triplicate. bTGA traces and NMR spectra for each sample were measured separately three times. cICP-MS and ICP-AES measurements: a digested NP sample was subjected to dilution in triplicate followed by separate measurements.

for sample 22E and ICP-AES measurements for sample 17A. The ICP-AES measurement offers the advantage of detecting simultaneously both the 89Y content of the NP and the 31P contribution of the phosphate on the ligand.16 This ratiometric method reduces the errors that can arise from sample handling, including washing and purification steps. Since each 31P corresponds to one PEG-OPO2H3 ligand, we could calculate the ligand density from the ratio of phosphorus to yttrium counts. These values for mPEG750 (1.1 ± 0.2 nm−2) and mPEG2000 (1.1 ± 0.1 nm−2) on 17A are somewhat smaller than values determined by TGA. In contrast, mPEG5000 on 17A (0.18 ± 0.09 nm−2) and mPEG2000 on 22E (2.3 ± 0.5 nm−2), the PEG densities are within experimental error of values determined by TGA. As a third independent measurement, the PEGylated NPs were examined by 1H NMR in D2O, using 2,2-dimethyl malonic acid (DMMA) as an internal standard. The PEG ligand densities were determined by comparing the integrations of the methoxy peak of mPEG (s, 3.38 ppm) with that of the two methyl groups (s, 1.31 ppm) of DMMA. In this way, we calculated values of ligand densities for sample 17A of mPEG750 (0.83 ± 0.06 nm−2), mPEG2000 (1.7 ± 0.1 nm−2), and mPEG5000 (0.27 ± 0.01 nm−2). For sample 22E, we found a value of 1.9 ± 0.5 nm−2 for mPEG2000. (Figure S8, Supporting Information). The coverage densities of PEG ligands determined by each of these measurements are collected in Table 2. A few more comments are in order about the NMR spectra in Figure 5. The peaks for the PEG ligands on the NPs are sharp and well-defined. Even the peak at 4.2 ppm for the CH2 group adjacent to the phosphate can be seen. One can infer that the PEG chains bound to the NP surface remain highly mobile. As a consequence, it would be difficult to distinguish between free and bound PEG ligands by 1D proton NMR. One can also observe that the methyl peaks of the DMMA internal standard is broadened in the NMR spectra. We surmise that DMMA may undergo some ligand exchange or adsorption onto the NPs due to its two carboxylic acid groups. To put our ligand density values in context for the mPEGphosphate ligands, we note that the expected area can be approximated by the area occupied by phosphate head groups in a lipid bilayer, ca. 0.34 nm2. This area corresponds to a coverage density of 1.6 phosphates/nm2. The ligand density of both PEG750 and PEG2000 are consistent with this theoretical limit, whereas the packing of high molecular weight PEG5000phophate is substantially reduced, presumably due to the steric hindrance of the polymer chains. The main conclusion from the results presented in Tables 1 and 2 is that ligand surface coverage values determined independently on the same sample by different methods are largely in agreement. There are some differences that remain unexplained, for example, the much lower value for mPEG750 on sample 17A determined by NMR compared to that by TGA and

Figure 5. 1H NMR spectra of NaYF4 NPs capped with methoxyPEG monophosphate with the following molecular weights: (A) 750, (B) 2000, and (C) 5000. NMR spectra were taken in D2O (*) containing a known amount of dimethyl malonic acid as an internal standard. Peak assignments are shown on the structures included in the spectrum. Although methoxyPEG monophosphate is drawn as the fully protonated monophosphate, its degree of protonation as bound to the NP surface is unknown.

ICP-AES. Also important is the result from Figure 1 that the surface coverage of NaYF4 NPs, at least for oleates, is sensitive to the protocol used to separate and purify the NPs prior to the analysis. We have some results suggesting a similar sensitivity for PEG-ligand coverage of NaLnF4 NPs. In our previous work, we reported much higher ligand densities for mPEG750-OPO3H2 and mPEG2000-OPO3H2 bound to NaGdF4 NPs determined by TGA analysis.32 Those ligand-exchanged NP samples were purified by dialysis rather than spin filtration. Dialysis seems to be much less effective than spin filtration at removing excess PEG ligands from a NP sample. Analysis of mPEG-Monophosphate and mPEG-Tetraphosphonate Ligands on NaTbF4 NPs. We recently reported the synthesis of a lysine-based mPEG2000-tetraphosphonate (mPEG2000-(PO3H2)4) ligands for lanthanide NPs.25 NaLnF4 NPs capped with this ligand showed much better colloidal stability in phosphate-containing buffers including PBS compared to that of NPs capped with mPEG-OPO3H2 ligands. We would like to better understand the colloidal stability of these NPs by comparing the surface coverage of both ligands. We used TGA to characterize mPEG2000-Lys(PO3H2)4 on uniform NaTbF4 NPs (dTEM = 10.9 ± 0.4 nm, CV < 4%) and compared the ligand density to mPEG2000-OPO3H2 bound to the same particles as shown in Figure 6. To minimize the effects of the sample purification protocol, the ligand exchange and sample washing (by spin filter) steps followed those described above for NaYF4 samples 17A and 22E. The NaTbF4 NPs were H


Article


Figure 6. TEM image (A) and size distribution (B) histogram of NaTbF4 NPs (dTEM = 10.9 ± 0.4 nm) used for ligand characterization by TGA. TGA measurements show the mass loss from these NaTbF4 NPs (black dashed lines) and of the ligand itself (red lines). (C) As-prepared oleate-coated NPs; (D), mPEG2000-OPO3H2-coated NPs; and (E) mPEG2000-Lys(PO3H2)4-coated NPs. The chemical structures of the ligands are displayed to the right of each TGA trace.

PEG chains form a water-swollen corona around each NP, and the corona thickness L can be inferred by comparing the hydrodynamic diameter dh of the NPs as determined by DLS with the hard-core NP diameter dTEM determined by TEM. The relationship between these quantities is depicted in Figure 7. For

synthesized under the same reaction conditions as for NaYF4 NPs except with different amounts of OA and ODE (here 1:1 by volume) in the reaction mixture and a longer reaction time (90 min). After the NPs were washed by two sedimentation− redispersion cycles with ethanol/hexanes, the NPs were ligand exchanged with mPEG2000-Lys(PO3H2)4 or mPEG2000OPO3H2 using the same procedure as that described for the NaYF4 NPs. For the as-synthesized OA-stabilized NPs, the TGA mass loss was 14.0%, corresponding to 3.4 OA/nm2. This value is significantly smaller than the value (8.2 OA/nm2) found for sample 17A, the 17 nm OA-coated NaYF4 NPs. After ligand exchange with mPEG2000-OPO3H2, we found 1.5 ligands/nm2, corresponding to a footprint of 0.67 nm2. The surface coverage is comparable to that (1.7 nm−2) found for this ligand on the 17 nm NaYF4 NPs. For the tetraphosphonate ligand, we found 1.8 ligands/nm2, corresponding to a footprint of 0.56 nm2. The footprint of the multidentate ligand with its four phosphonate groups is essentially identical to that of the monodentate mPEG2000-OPO3H2. As mentioned above, the limiting density of phosphate groups is about 1.6 nm−2,33 which suggests that on average fewer than two of the four phosphonates on the tetraphosphonate ligand are bound to the surface of the NP at a given time. If this is the case, then the origin of the enhanced colloidal stability of NPs bearing this tetradentate ligand may be due to the dynamic nature of the binding, in which proximity effects help the tetraphosphonate ligand compete with external phosphate and minimize ligand displacement. Dimensions of the PEG Corona. As mentioned above, consistent results were obtained by TGA, NMR, and ICP-AES for the PEG-chain densities on NaYF4 NP sample 17A after ligand exchange with three different lengths of mPEG-OPO2H3. We found 1.5 ligands/nm2 for mPEG750-OPO2H3 and mPEG2000-OPO2H3, and 0.2 ligands/nm2 for mPEG5000OPO2H3. For sample 22E (dTEM = 22 nm) after ligand exchange with mPEG2000-OPO2H3, we found ca. 2 ligands/nm2. The

Figure 7. Determination of the corona thickness L from the difference between the TEM diameter of the NP and the hydrodynamic diameter of the PEGylated NP.

sample 17A, we calculate L values of 10, 12, and 16 nm for mPEG750, mPEG2000, and mPEG5000, respectively. For sample 23E, we obtain an L value of 9.3 nm for mPEG2000 (dh = 41.3 nm, dTEM = 22.8 nm). Note that L values calculated in this way are somewhat exaggerated in magnitude because dh is the z-average hydrodynamic radius, which heavily weights the largest contributors in the sample, whereas dTEM is the numberaverage core diameter. It is instructive to compare the spacing of the PEG ligands on the NP surface with the dimensions of the PEG chains as described, for example, by the root-mean-squared end-to-end distance Rf (the Flory radius). The theory of polymer brushes34 predicts that if the distance D between the anchor points of adjacent chains is significantly smaller than Rf or smaller than twice the radius of gyration (Rg), corona chain repulsion will lead I


Article


NaYF4 NPs and of PEG content after ligand exchange with three different mPEG-OPO3H2 chain lengths gave results that were consistent for each sample. Thus, the values determined for the ligand densities on each sample can be considered reliable. Nevertheless, we found a significant variability in surface coverage associated with the details of the sample isolation and purification protocol, suggesting that there may not be a specific stoichiometry associated with carboxyl groups of oleate or phosphate groups of the PEG ligand interacting with metal ions at the NP surface. Another issue that needs further investigation in the future is that the oleate-coated NPs showed surface coverages (5.5 to 8 OA/nm2) larger than commonly accepted values for the maximum ligand density on a two-dimensional surface (∼5 OA nm−2).40 The absence of olefinic proton resonances in 1H NMR measurements on the oleate coated NaYF4 NPs, however, is consistent with strongly immobilized oleate groups, suggesting that all of the OA molecules are tightly associated with the NP. For PEGylated NPs coated with mPEG750 and mPEG2000, the coverage densities were comparable (∼1.1 to 1.7 nm−2), whereas the corona repulsion for the mPEG5000 chains limited the surface coverage to 0.2 nm−2. These packing densities, particularly for mPEG2000 and mPEG5000, are consistent with overlapping solvent-swollen corona chains that promote chain stretching normal to the particle surface. This is a necessary prerequisite to minimize protein adsorption on the PEGylated NPs employed for in vitro and in vivo studies. Another important factor is colloidal stability in buffers and physiological media. NaLnF4 NPs stabilized with PEG-monophosphates fail this test since they precipitate in media containing inorganic phosphate. We have previously shown that 10 nm NaTbF4 NPs stabilized with mPEG2000-Lys(PO3H2)4, a tetraphosphonate ligand, exhibits good colloidal stability in PBS buffer. In this work, we have shown that the high surface coverage of this ligand corresponds to a very small footprint (0.56 nm2) on the surface of NaTbF4 NPs, essentially identical to that of the monodentate mPEG2000-OPO3H2. The value of 1.8 ligands/nm2 found for the tetradentate ligand is close to the limiting density of phosphate groups (1.6 nm−2)33 on a two-dimensional surface. This result suggests that on average fewer than two of the four phosphonates on the tetraphosphonate ligand are bound to the surface of the NP at a given time. We would also like to emphasize that the oleate surface coverages achieved in our experiments were very sensitive to the details of the protocols employed during separation of the NPs from the synthesis mixture and the subsequent purification of the NPs. Surface coverage with PEG-monophosphates also appears to be sensitive to the ligand exchange protocol including the methodology used to remove excess ligands from the NPs. Our results emphasize the importance of reporting this level of characterization, particularly for polymer-modified NaLnF4 NPs intended for in vitro and in vivo studies.

to an increase in chain dimensions normal to the surface and increase the magnitude of L. For long polymer chains, scaling laws describe the relationship between D, Rg, and L, both for polymers anchored to planar surfaces and to spherical polymer brushes. These relationships are unlikely to apply to chains as short as PEG750, but there is some evidence that they can apply at high surface coverage to PEG2000 and PEG5000.35 We estimate the distance between polymer chains on the NP surface by assuming that the area occupied by each ligand is a circle with a diameter equal to the distance between adjacent chains. On the basis of this approximation, the distances among PEG750, PEG2000, and PEG5000 ligands are 1.1, 1.4, and 2.5 nm, respectively. Values of the Flory radius can be estimated from the scaling relationship Rf = aNυ, and values of a = 0.35 nm and ν = 3/5 are considered appropriate for PEG chains in water.35,36 In this way, we calculate values of Rf = 2, 3.4, and 6 nm, respectively, for PEG750, PEG2000, and PEG5000. For PEG2000 and PEG5000, the distance between anchor points is smaller than the characteristic chain dimensions, suggesting chain stretching in the corona. As summarized in Table 3, the L values calculated from the NP core diameter and the hydrodynamic diameter are much larger than the estimated values of Rf. Table 3. Characteristic Diameters, Interchain Spacing, and Corona Thickness for PEG Ligands on NaYF4 NPs ligand-NP mPEG75017A mPEG200017A mPEG500017A mPEG200023E

dTEM (nm)

dh (nm)

PDI

D (nm)

Rf (nm)a

L (nm)b

17

37, 119c

0.27

1.1

2

10

17

41.6 ± 0.5d

0.26

1.4

3.4

12

17

48.9 ± 0.3d

0.27

2.5

6

16

23

41.3 ± 0.2d

0.16

2

3.4

9

a Calculated with the expression Rf = aNυ, with a = 0.35 nm and ν = 3/ 5. bCalculated with the expression L = (dh − dTEM)/2. cThese values correspond to peaks in the bimodal CONTIN plot, with the larger peak indicating the presence of aggregates in the sample. dz-Average values calculated from the first cumulant in a cumulant analysis.

The corona thicknesses (brush heights) determined for mPEG2000 and mPEG5000 on NaYF4 NPs are comparable to those obtained for other types of NPs such as iron oxide (dTEM = 12 nm, PEG2000, L = 13 nm)37 and gold NPs (dTEM = 17 nm, PEG5000, L = 11.7 nm).38 It is curious that the calculated value of L for PEG750 on NaYF4 NPs (10 nm) exceeds that of the fully extended chain length of PEG750 (17 EO units, 4.5 nm). We note, however, that a similar large value for the brush height of PEG750 on a gold NP sample (dTEM = 15 nm, L = 9 nm) has also been reported.39 While we have no unique explanation for this result, one can imagine some possibilities to explain this result. For example, aggregates in solution may contribute to the peak position in the CONTIN plot from which the value of dh was inferred. Alternatively, a few PEG chains associated with the corona that are not end-anchored on the NP surface may protrude into the aqueous phase and contribute to the hydrodynamic dimensions of the NP.

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ASSOCIATED CONTENT

S Supporting Information *

Setup for NaTbF4 NP synthesis; synthesis of mPEG5000OPO3H2; characterization of the NaYF4 and NaTbF4 NPs by TEM and XRD; and NMR spectra of the pure ligands and the internal standards; 1H NMR T1 relaxation times of protons of the internal standards and ligands; thermogravimetric analysis of PEGylated NaYF4 NP sample 17A; and ICP-AES instrument parameters and operation conditions. The Supporting Informa-

■

CONCLUSIONS Independent measurements by TGA, 1H NMR, and ICP-MS or ICP-AES of surface coverage of oleate groups on as-synthesized J


Article


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tion is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.5b02190.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 416 978 6495. Fax: +1 416 978 0541. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the NSERC Canada and DVS Sciences (now Fluidigm Canada, Markham, ON), the Office of AIDS Research, National Institutes of Health, and the Province of Ontario for their support of this research. We acknowledge Professor Frank C. J. M. van Veggel and Dr. Noah Johnson from the University of Victoria, British Columbia for assisting L.T. in the synthesis of NaYF4 nanoparticles. We also thank Dr. D. Burns for his assistance with the NMR experiments. We acknowledge the Canadian Foundation for Innovation, project number 19119, and the Ontario Research Fund for funding of the Centre for Spectroscopic Investigation of Complex Organic Molecules and Polymers.

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Quantification of Surface Ligands on NaYF4 Nanoparticles by Three

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