We are interested in developing lanthanide nanoparticles (NPs) as high sensitivity tagging reagents for antibodies to analyze cells by mass cytometry (MC). Two key prerequisites for this application are that the NPs have to be colloidally stable in phosphate-containing buffers and the free NPs must have very low levels of nonspecific binding to cells. These are the issues we address here. We describe the synthesis of 30 nm diameter NaYF4:Yb,Er nanoparticles, their transfer to aqueous solution via citrate exchange, and their encapsulation in liposomes to minimize their interaction with live cells. The lipid coating consisted of a 2:2:1 mol ratio mixture of dioleoylphosphatidyl choline (DOPC), egg sphingomyelin (ESM), and ovine cholesterol (Chol), referred to as DEC221. Since encapsulating 30 nm NPs in liposomes is an unprecedented challenge, we added varying amounts of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG-2000] (mPEG2K-DSPE) to the lipid formulation, both to promote curvature of the lipid coating and to use the polyethylene glycol (PEG) chains to impart stealth and minimize interaction with cells. We succeeded in coating individual NPs with the lipid bilayer and showed that, after coating, the NPs were colloidally stable in PBS buffer for up to one month. We used MC to measure nonspecific binding of the lipid-coated NPs to three different suspension cell lines, Ramos, THP-1, and KG1a cells. For dosages of 50, 100, and 1000 NPs/cell, the measured signals were barely above background. For dosages of 10 000 and 30 000 NPs/cell, nonspecific binding levels were on the order of 10–15 NPs per cell, less than 0.1% of the applied dose. Dopant ions such as Yb also provide a measurable signal, indicating that NaYF4 NPs can serve as a useful host matrix for different lanthanide dopants for multiparameter experiments. These are very encouraging results for future experiments in which specific antibodies will be incorporated into the lipid coating.
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 NaYF 4 and NaTbF 4 NPs, as well as the surface coverage after ligand exchange with three methoxyPEG-monophosphates with M n = 750, 2000, and 5000 Da. For NaYF 4 , we obtained consistent results for both oleates and PEGs by three independent methods (TGA, 1 H 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 NaTbF 4 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.
The range of properties available in the lanthanide series has inspired research into the use of lanthanide nanoparticles for numerous applications. We aim to use NaLnF(4) nanoparticles for isotopic tags in mass cytometry. This application requires nanoparticles of narrow size distribution, diameters preferably less than 15 nm, and robust surface chemistry to avoid nonspecific interactions and to facilitate bioconjugation. Nanoparticles (NaHoF(4), NaEuF(4), NaGdF(4), and NaTbF(4)) were synthesized with diameters from 9 to 11 nm with oleic acid surface stabilization. The surface ligands were replaced by a series of mono-, di-, and tetraphosphonate PEG ligands, whose synthesis is reported here. The colloidal stability of the resulting particles was monitored over a range of pH values and in phosphate containing solutions. All of the PEG-phosphonate ligands were found to produce non-aggregated colloidally stable suspensions of the nanoparticles in water as judged by DLS and TEM measurements. However, in more aggressive solutions, at high pH and in phosphate buffers, the mono- and diphosphonate PEG ligands did not stabilize the particles and aggregation as well as flocculation was observed. However, the tetraphosphonate ligand was able to stabilize the particles at high pH and in phosphate buffers for extended periods of time.
Developing surface coatings for NaLnF4 nanoparticles (NPs) that provide long-term stability in solutions containing competitive ions such as phosphate remains challenging. An amine-functional polyamidoamine tetraphosphonate (NH2-PAMAM-4P) as a multidentate ligand for these NPs has been synthesized and characterized as a ligand for the surface of NaGdF4 and NaTbF4 nanoparticles. A two-step ligand exchange protocol was developed for introduction of the NH2-PAMAM-4P ligand on oleate-capped NaLnF4 NPs. The NPs were first treated with methoxy-poly(ethylene glycol)-monophosphoric acid (Mn = 750) in tetrahydrofuran. The mPEG750-OPO3-capped NPs were stable colloidal solutions in water, where they could be ligand-exchanged with NH2-PAMAM-4P. The surface amine groups on the NPs were available for derivatization to attach methoxy-PEG (Mn = 2000) and biotin-terminated PEG (Mn = 2000) chains. The surface coverage of ligands on the NPs was examined by thermal gravimetric analysis, and by a HABA analysis for biotin-containing NPs. Colloidal stability of the NPs was examined by dynamic light scattering. NaGdF4 and NaTbF4 NPs capped with mPEG2000–PAMAM-4P showed colloidal stability in DI water and in phosphate buffer (10 mM, pH 7.4). A direct comparison with NaTbF4 NPs capped with a mPEG2000-lysine-based tetradentate ligand that we reported previously (22906305Langmuir2012281286112870) showed that both ligands provided long-term stability in phosphate buffer, but that the lysine-based ligand provided better stability in phosphate-buffered saline.
Over the past decade, there have been extensive developments in the field of lanthanide-based nanoparticles (NPs). Most studies have focused on the application of upconverting NaYF4-based NPs for deep tissue imaging and paramagnetic NaGdF4 NPs for MRI. Current applications for the remaining members of the lanthanide series are rather limited. Recently, a novel bioanalytical technique known as mass cytometry (MC) has been developed which can benefit from the entire lanthanide series of NPs. MC is a high-throughput multiparametric cell-by-cell analysis technique based on atomic mass spectrometry that uses antibodies labeled with metal isotopes for biomarker detection. NaLnF4 NPs offer the promise of high sensitivity coupled with multiparameter detection, provided that NPs can be synthesized with a narrow size distribution. Here we describe the synthesis of six members of this NP family (NaSmF4, NaEuF4, NaGdF4, NaTbF4, NaDyF4, NaHoF4) with the appropriate size (5–30 nm) and size distribution (CV < 5%) for MC. We employed the coprecipitation method developed by Li and Zhang [Nanotechnology 2008, 19, 345606], and for each member of this series, we examined the heating rate, final reaction temperature, and composition of the reaction mixture in an attempt to optimize the synthesis. For each of the six NaLnF4, in the range of the target sizes, we were able to identify “sweet spots” in the reaction conditions to obtain NPs with a narrow size distribution. In addition, we investigated the oleate surface coverage of the NPs and the effect of long-term storage (2 years) on the colloidal stability of the NPs. Finally, NaTbF4 NPs were rendered hydrophilic via lipid encapsulation and tested for nonspecific binding with KG1a and Ramos cells by mass cytometry.
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