Liposome-Encapsulated NaLnF4 Nanoparticles for Mass Cytometry

May 11, 2017 - Liposome-Encapsulated NaLnF4 Nanoparticles for Mass Cytometry: Evaluating Nonspecific Binding to Cells. Jothirmayanantham Pichaandi,...
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Liposome-Encapsulated NaLnF4 Nanoparticles for Mass Cytometry: Evaluating Nonspecific Binding to Cells Jothirmayanantham Pichaandi,†,# Lemuel Tong,†,# Alexandre Bouzekri,‡ Qing Yu,† Olga Ornatsky,‡ Vladimir Baranov,‡ and Mitchell A. Winnik*,† †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3HS, Canada Fluidigm Canada Inc., 1380 Rodick Street, Markham, Ontario L3R 4G5, Canada

‡

S Supporting Information *

ABSTRACT: 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 1,2distearoyl-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.

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INTRODUCTION Identifying multiple cellular biomarkers in a high throughput single cell assay is an important task in contemporary biology and medicine. Flow cytometry is the most commonly used technique and employs monoclonal antibodies (mAbs) labeled with fluorescent dyes for biomarker recognition.1 The width of fluorescence emission bands leading to overlap and the need for compensation limits the number of biomarkers that can be detected conveniently on individual cells to relatively small numbers, on the order of 5 to 10, although an 18-plex assay has been reported in the literature.2 Over the past decade, the development of mass cytometry (MC) has greatly increased the number of biomarkers per cell that can be detected at high throughput and also allows quantitative determination of biomarker expression.3 In MC, mAbs are labeled with heavy metal isotopes, most commonly lanthanide isotopes, and cells are analyzed individually but stochastically by inductively coupled plasma mass spectrometry (ICP-MS) with time-offlight detection.4 Because of single mass resolution over the range of m/e 80 to 220 amu, signal overlap is minimal and the © 2017 American Chemical Society

number of biomarkers that can be detected from an individual cell is limited only by the number of different accessible isotopes that one can attach to Abs. MC enables simultaneous detection of 40+ biomarkers on a routine basis, and in principle, simultaneous detection of up to 100 biomarkers per cell is possible.5−7 The signal generated in an ICP-MS measurement increases linearly with the number of copies of each metal isotope attached to an affinity reagent. As a way of generating a strong signal, metal-chelating polymers with multiple chelating groups per polymer are attached to each Ab.8−10 Each Ab carries 150 to 250 copies of isotope,11,12 and the technique is very effective at detecting biomarker expression at levels of 104 to 107 biomarkers per cell.11,13 While there are no reports establishing a lower limit for biomarker detection by MC with metalchelating polymer reagents, we encountered a sensitivity issue Received: April 3, 2017 Revised: May 11, 2017 Published: June 5, 2017 4980

DOI: 10.1021/acs.chemmater.7b01339 Chem. Mater. 2017, 29, 4980−4990

Article

Chemistry of Materials when attempting to detect much smaller numbers of biomarkers in a model system. The model system consisted of 0.7 μm diameter colloidal hydrogels with small numbers of streptavidin (SAv), as a model biomarker, bound to the surface. Here, we found that treatment of the hydrogel with a biotinylated metal-chelating polymer carrying 50 159Tb3+ ions led to no detectable signal by MC.14 Lanthanide nanoparticles (NPs) of the general structure NaLnF4 are very attractive as potential high sensitivity reagents for MC. For example, a NaHoF4 NP with an effective diameter of 10 nm will have about 8000 Ho3+ ions and a 20 nm NP will have ca. 50 000 Ho3+ ions per NP. When we treated the colloidal hydrogel sample with biotinylated 13 nm NaHoF4 NPs (15 000 165Ho/ NP), we could detect binding of 100 NPs per hydrogel. While this is a primitive experiment, with no attempt to optimize the surface coating of the NPs, it establishes the enhanced sensitivity of NP reagents if other problems like colloidal instability of NPs in buffers and nonspecific binding associated with NPs can be overcome. To serve as effective high-sensitivity reagents for single cell analysis by MC, NaLnF4 NPs have to meet four essential criteria:

Lipid coating is an alternative to ligand exchange for surface modification of NPs. Oleate-coated NPs can be coated with a lipid monolayer.25 We used a thin-film hydration method to coat 10 nm diameter NaTbF4 NPs with a 70:30 (by mole) mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG-2000] (mPEG2K-DSPE).28 While these NPs had only limited colloidal stability in phosphate buffer, they had sufficient stability so that we could examine their interaction with KG1a and Ramos cells by MC following incubation in PBS containing 0.5 wt % bovine serum albumin (BSA). We found low levels of nonspecific binding (NSB) for live cell suspensions containing up to 50 NPs/cell and for cells that were fixed prior to being treated with the lipid-coated NPs at a dosage of 500 NPs/cell. In contrast, at a dosage of 500 NPs/ cell, the NSB for live cells was almost 10% of the applied dose. Thus, there are two deficiencies that need to be overcome in the development of NP reagents for MC: They have insufficient colloidal stability in phosphate buffer, and the nonspecific binding background will be too high in experiments on live cell suspensions that will likely be carried out at 1000− 10 000 NPs per cell. Thus, alternative coating approaches are needed. In this paper, we describe an approach in which the NPs are encapsulated in phospholipid bilayer vesicles (liposomes) modified to minimize cell uptake. Liposomes are attractive because they are colloidally stable in physiological media and have been used as carriers for both hydrophilic and hydrophobic agents. There is a broad interest in liposomal therapeutics,29 and applications include the encapsulation of small molecule drugs such as doxorubicin,30,31 paclitaxel,32,33 and cisplatin,34,35 as well as larger molecules such as siRNA36 and DNA.37 The size and shape of liposomes are influenced by the lipid composition and the physicochemical properties of the polar head groups. In addition, polymers such as polyethylene glycol (PEG) can be grafted onto lipids to improve their pharmacokinetics for in vivo applications, with longer circulation times and lower clearance by the mononuclear phagocytic system (MPS).38 In spite of these attractive features, liposomes have not been widely used to encapsulate NPs. The most difficult problem has to do with vesicle size and radius of curvature. It is difficult to encapsulate individual nanoparticles with sizes on the order of 30 nm or less. For example, attempts to encapsulate maghemite (Fe2O3) NPs with d ∼ 15 nm resulted in multiple particles inside each liposome.39−41 There is a second technical problem for NaLnF4 NPs: They have to be dispersed in aqueous media prior to the encapsulation by the liposomes. There are a few reports in the literature describing lipid bilayer coating of individual NPs. One set of examples involves hydrophilic silica NPs with diameter d ∼ 50−60 nm.42−44 Another report describes the coating of individual calcium phosphate NPs for drug delivery.45 The most common lipids used for bilayer encapsulation of NPs are dimyristoylphosphatidyl choline (DMPC), cholesterol, mPEG2K-DSPE, and functional derivatives of PEG2K-DSPE. The relative amounts of these lipids are varied in order to modify liposome size. Recent reports by Walker and co-workers48,49 describe a lipid mixture consisting of a 2:2:1 mol ratio of dioleoylphosphatidyl choline (DOPC), egg sphingomyelin (ESM), and ovine cholesterol (Chol), referred to as DEC221. This mixture was examined as a model cell membrane on mica46 and gold surfaces.47 In addition, the Walker group was able to

1. Uniform size with diameters in the range of 5−30 nm and Cv less than 5%. 2. Surface modification that renders the NPs colloidally stable in physiological buffers and in the presence of biomolecules. 3. A surface coating that provides effective stealth to minimize nonspecific binding to cells. 4. A surface coating with appropriate functional groups for attachment of Abs or other affinity agents. There has been substantial progress in recent years in the synthesis of NaLnF4 NPs, particularly Yb, Er doped NaYF4 and other doped NaYF 4 NPs 15 that exhibit upconversion luminescence (UCNPs), as well as in NaGdF416 and NaDyF417 NPs for magnetic resonance imaging (MRI) applications. We have reported the synthesis of a series of NaLnF4 NPs (Sm to Ho) in this size range with a sufficiently narrow size distribution (Cv(diameter) ≤ 5%) that satisfies the current requirements for application to MC.18 These assynthesized NPs are coated with oleic acid (OA) molecules rendering them hydrophobic. For biomedical and bioanalytical applications, the surface of the NPs has to be modified to provide colloidal stability in water, in phosphate-containing buffers, and in physiological media. Optimizing the surface of these NPs to meet these stringent requirements is a significant challenge. One initial approach to surface modification of oleate-coated NaLnF4 NPs involved ligand exchange.19−21 Polyethylene glycol (PEG) derivatives with a phosphate at one end displace oleate from the surface of the NPs allowing transfer of the NPs to water. These particles, however, flocculate in phosphate containing media unless kept in the presence of excess PEGOPO3H ligand.22 Multidentate PEG based ligands containing 2 or 4 phosphonate groups are more effective at providing stability in PBS buffer but lead to other problems, such as particle etching, nonspecific interactions with cells, and synthetic difficulties in modifying the reagents to attach antibodies.20−24 Another approach is to encapsulate the Ln NPs with amphiphilic polymers such as poly(maleic anhydridealt-1-octadecene)-co-poly(ethylene glycol) and octylamine (OA)-grafted poly(acrylic acid) (PAA).25−27 4981

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lanthanide elements (La to Lu including yttrium, Y). In order to obtain a calibration curve, we prepared 0.1, 1, and 10 ppb standard solutions. All ICP-MS measurements were carried out in triplicate, and the final ppb values were averaged from the measurements. Mass Cytometry. MC experiments were conducted with a Helios instrument at Fluidigm Canada (Markham, ON). Cells were dispersed in 100 μL of DI water, and an aliquot (10−20 μL) of the standard reference bead stock solution (EQ-4 beads) was added to the cells. The data were collected in FCS3.0 file format and processed by FlowJo software. The number of NaYF4:Yb,Er NPs per cell was calculated using the mass cytometry transmission coefficients for Y (3.63 × 10−5) and the number of Y atoms (1.4 × 105) per 30 nm of NaYF4:Yb,Er UCNP. DEC221 Bilayer Lipid Encapsulation. The preparation of the DECC221 lipid mixture and NP encapsulation was adapted from a procedure developed by Ip et al.48,49 for encapsulating Au NPs. The lipid films were first prepared in 7 mL scintillation vials. The DEC221 lipid mixtures were prepared on a 20 mg scale and then divided into 10 aliquots with 2 mg (3.0 μmol) of lipids per vial. The DEC221 mixture contained DOPC (9.30 mg, 11.8 μmol), ESM (8.41 mg, 11.8 μmol), and Chol (2.29 mg, 5.9 μmol) in a 2:2:1 molar ratio. The lipids were dissolved in a CHCl3 (3 mL)/MeOH (1 mL) mixture; then, aliquots (2 mg solids) were placed in glass vials and dried into thin films under an Ar (g) flow for 1 h. Residual solvent was removed under vacuum at room temperature overnight. The vials were purged with Ar, capped, sealed with paraffin, and stored at −20 °C for future use. As a reference, a sample of DEC221 liposomes without DSPE-PEG was prepared in water by hydrating a lipid film sample (1 mL of water, heated at 50 °C in an aluminum heating block for 30 min) and then sonicated at 50 °C for 1 h until the solution became clear. The solution was then stored overnight at 4 °C. This lipid suspension was processed using a lipid extruder (c.f., Figure S3) by passing the sample back-and-forth 13 times through a 100 nm polycarbonate filter while maintaining the temperature at 50 °C. After cooling the sample, the liposomes were characterized by DLS at 25 °C (Figure S4), with a hydrodynamic diameter (dh) of 43.5 ± 0.25 nm (PDI = 0.25 ± 0.01). This sample was stored at 4 °C. We used this sample to estimate the number of liposomes formed from 2 mg of DEC221 lipids under our sample preparation conditions. To begin, we calculate the number of lipids (N) per liposome with eq 1, which assumes that the liposomes are uniform spherical unilamellar vesicles.

encapsulate 60 nm gold NPs with a bilayer of this lipid mixture.48,49 These lipid-coated gold NPs were prepared by thin-film hydration using either water or phosphate buffered saline to form multilamellar vesicles (MLVs), which were then sonicated to form unilamellar vesicles (ULVs). These transparent solutions showed a surface plasmon resonance band in the UV−vis absorption spectrum consistent with no aggregation of the gold nanoparticles. Direct evidence for the formation of individual NPs with a lipid coating was obtained from transmission electron microscopy (TEM) images and dynamic light scattering (DLS) measurements. Note that our goal of coating NPs with lipids and exposing them to cells at 4 °C to minimize uptake or binding, as a step toward developing them as tagging reagents for mass cytometry, is very different from the approach recently reported by the Irvine group at MIT.50 They used mass cytometry to evaluate uptake of 3 nm gold nanoparticles, with various surface coatings, by cells and by tissues under physiological conditions. In the experiments described here, we examine encapsulation of NaYF4:Yb,Er NPs with d = 30 nm with a DEC221 mixture of lipids to which varying amounts of mPEG2K-DSPE (1, 2, 3, and 6 mol %) were added. The colloidal stability of the coated NPs in water and in physiological buffers was monitored by DLS, and the overall size of the modified NPs was characterized by both DLS and TEM. Finally, the liposome-encapsulated NPs were examined for their interaction with different cell lines. For dosages ranging from 50 to 30 000 NPs/cell, we found low nonspecific binding levels of less than 0.2%.

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EXPERIMENTAL SECTION

Materials. The following were purchased from Sigma-Aldrich and used without further purification: citric acid (>99.5%), sodium citrate dihydrate (>99%), sodium chloride, Tris, HEPES, and phosphate buffered saline (PBS). The following were purchased from Avanti Polar Lipids, Inc. and used without further purification: 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (ESM), ovine cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (mPEG2K-DSPE). Maxpar Ab reagents and EQ Four element calibration beads were provided by Fluidigm Canada. The synthesis of oleate-coated NaYF4:Yb,Er UCNPs and one example of transfer to citrate buffer (dh = 35.6 ± 0.4 nm, dTEM = 23.3 ± 0.6 nm, Figures S1 and S2) are described in the Supporting Information. Instrumentation. All lipid vesicle samples were prepared with a Branson 2800 bath sonicator (110 W) at 50 °C for 1 h. Dynamic light scattering (DLS) measurements were performed with a Malvern Zetasizer Nano ZS at 25 °C with a 173° backscatter measurement angle. The samples were generally filtered through a nylon syringe filter (0.2 μm) before the measurements. CONTIN plots were obtained by analysis of representative autocorrelation decay profiles using instrument software. Apparent z-average hydrodynamic diameters, dh, and corresponding PDI values were calculated via a cumulant analysis with instrument software. Results are reported as the average and standard deviations of three replicate measurements. The Zetasizer was also used to measure the electrophoretic motilities of the samples. Transmission electron microscopy (TEM) measurements were carried out using Hitachi HT7700. The samples were drop-cast on to carbon-Formvar grids and then dried in air before the measurements. As citrate coated NPs are hydrophilic in nature, the grids were pretreated with Triton-X305 (1 wt %) to render the surfaces hydrophilic. ICP-MS analysis employed an ELAN 9000 instrument. The citrate- and liposome-encapsulated NPs were dissolved in 2 vol % HNO3. The samples were diluted with 2 vol % HNO3 until ppb concentrations were obtained. Standard lanthanide solutions were prepared by a series of 10-fold dilutions of a stock solution (1000 mg/L, 2% HNO3, PerkinElmer) containing all of the

N=

4π(r 2 + (r − h)2 ) A

(1)

Taking the radius r of the liposomes to be half of the hydrodynamic diameter (dh = 43.5 ± 0.25 nm), h = 5 nm for the thickness of the lipid bilayer, and A = 0.72 nm2 for the area of the lipid headgroup for DOPC, we calculate ca. 1.3 × 104 lipids/liposome. Therefore, the number of liposomes in a 2 mg sample is ca. 1.2 × 1014. For NP encapsulation, the DEC221 lipid film was hydrated either in water or in a saline-containing buffer (1 mL) by heating at 50 °C and vortexing intermittently over 30 min. In parallel, a thin film of mPEG2K-DSPE (0.18 μmol, 6 mol % based on DEC221) was prepared and then hydrated in the same manner as the DEC221 sample. This sample gave a clear solution, which was combined with the turbid DEC221 lipid suspension, along with an aliquot of citrate coated NPs (3.8 × 1012 NPs in 60 μL of citrate buffer, corresponding to ca. 30 liposomes per NP). The resulting turbid mixture was sonicated at 50 °C for 1 h until the solution became clear and then stored overnight at 4 °C. Excess lipids were removed via two sedimentation−redispersion cycles (15 000g, 30 min, 4 °C). Afterward, the NP pellet was redispersed in DI water or saline buffer (1 mL). In order to reduce the size of the lipid coated NPs after centrifugation, the NPs were processed using a lipid extruder as described above (13 back-and-forth passages through a 100 nm polycarbonate filter at 50 °C) and then stored at 4 °C. The NP concentration was measured by ICP-MS. 4982

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10−4 (3900 Ho ions required to obtain 1 detector count). A single 60 nm NaHoF4 NP will generate ca. 350 counts. Yttrium on the other hand has a lower transmission coefficient (T = 3.6 × 10−5, 27 500 Y ions per count), and a 60 nm NaYF4 NP will generate ca. 50 counts. As a compromise between risking detector saturation and the challenges of coating smaller NPs, we focus on a target size of d ≈ 30 nm. These NPs will contain a total of ∼2 × 105 Ln ions/NP. Even at this size, lanthanides like holmium or terbium will still generate 40−50 counts per NP, whereas yttrium would generate 4 to 5 counts per NP. The synthesis of 30 nm yttrium based NPs has been well established. To promote the curvature required to coat these 30 nm NPs, we incorporate various amounts of mPEG2KDSPE into the lipid formulation. NaYF4 NPs have another advantage in that they can be doped with other lanthanides. At these doping levels, one can avoid detector saturation and the dopant elements can serve as tags to enable multiparameter experiments. To explore this option, we looked at ytterbium at a doping level of 20 mol percent in NaYF4 NPs. Ytterbium serves three purposes. First, it has six naturally occurring isotopes, which are present in varying levels, and second, when codoped with Er3+ ions (2 mol %), it can impart luminescence to the nanoparticle. Third, its upconverting luminescence is very useful as a monitoring tool while performing the lipid encapsulation. Of the seven naturally occurring isotopes of ytterbium, the most abundant is 174Yb (32%). The remaining six ytterbium isotopes are present in varying levels ranging from 0.3% to 20%. For a 30 nm NaYF4:Yb,Er NP, the ca. 12 000 174Yb3+ ions (T = 2.7 × 10−4) will contribute about 3 counts/NP. The actual doping level of 174 Yb in the NaYF4, Yb, Er nanoparticle is around 6 mol % of total lanthanide atoms. These additional isotopes can also be monitored and will provide us with data on the minimum level of doping required in yttrium based NPs to obtain an observable signal from the MC measurements. The lanthanide nanoparticles were synthesized using a procedure adapted from Li and Zhang.15 Details are presented in the Supporting Information. The as-synthesized NaYF4:Yb,Er NPs were stabilized by a surface layer of oleate molecules. For lipid encapsulation, the NPs must first be transferred to aqueous media. Following a protocol adapted from Capobianco and co-workers,52 we carried out a ligand exchange with citrate buffer (200 mM, pH 4) shown schematically in Figure S1. The exchange process was monitored using the upconversion luminescence from NaYF4:Yb,Er NPs. The NPs were purified by successive sedimentation−redispersion cycles using ethanol to precipitate the NPs and then vortexed in citrate buffer (200 mM, pH 7) for redispersion. A TEM image of these NPs is shown in Figure 1B below. The particles maintain their size (dTEM = 29 nm) and narrow size distribution after ligand exchange and transfer to water. The zeta potential (−43 mV, pH 7 citrate buffer, 2 mM) is consistent with a surface coating of citrate, and a DLS measurement (Figure 1D) shows a narrow size distribution with an apparent z-average hydrodynamic diameter larger (dh = 53 nm) than the size determined by TEM. Other samples of citrate-coated NPs (see Figure S2) also show values of dh larger than dTEM. Part of this difference reflects the fact that dh is a zaverage value, which places heavier weight on the larger components of the size distribution than dTEM, which is a number-average. The double layer surrounding the NPs in solution also adds to the magnitude of dh.

Cell Incubation Studies. Live KG1a, THP-1, and Ramos cells (2 × 106) were collected from growth media by centrifugation and resuspended in PBS with 0.5 wt % BSA. The cells were initially stained with a 103Rh-intercalator to identify dead cells in the sample followed by staining with a cocktail of Maxpar metal-conjugated antibodies for 30 min at room temperature. We used CD45-154Sm and CD20-147Sm for Ramos cells. In the case of KG1a cells, CD45-154Sm was added to identify the cells in addition to the DNA intercalator containing 191Ir and 193Ir. For THP-1 cells, CD45-154Sm was used to identify the cells in addition to the Ir-containing DNA intercalator. Antibody-stained cells were washed and resuspended in PBS (0.5% BSA, 100 μL). In the subsequent washing procedure, cells were pelleted by centrifugation (9400g, 2 min) and the supernatant was aspirated. Different concentrations of lipid-coated NaYF4:Yb,Er UCNPs (107 to 109 NPs in 100 μL) were incubated with cells (2 × 106) for 30 min at 4 °C for Ramos cells. Experiments with KG1a and THP-1 cells were examined both after 30 min and 1 h of incubation. In a set of control experiments, citrate-coated NaYF4:Yb,Er UCNPs were added to KG1a cells and examined after 30 min of incubation. The cells were then pelleted at 9400g for 2 min.51 The supernatant was aspirated, and the cells were washed three times with PBS to remove unbound NPs. The cells were fixed with paraformaldehyde (PFA) (1.6%) overnight at 4 °C and washed with cold (4 °C) PBS. The fixed cells were then incubated for 1 h at RT with the Ir-intercalator. Cells were washed once more with cold (4 °C) PBS to remove excess intercalator followed by suspension in deionized water in preparation for MC measurements. FlowJo Data Analysis. After each mass cytometry measurement, the .fcs raw data files were normalized against the bead standards. The processed data was then analyzed using FlowJo software (v.10.0). The gating process is demonstrated in the following example. The gating process for the Ab−NP conjugates with KG1a cells at the 1000 NP/ cell dosage is presented in Figure S9. In this figure, the gating was performed sequentially from part A to E. In part A, cells were separated from the calibration beads using the 153Eu signal. The calibration beads were used as a standard for normalizing the signals and to adjust the signals to the standard lanthanide concentrations. In part B, the live cells were gated from the dead cells by gating the 103Rh and 193Ir signals. 103Rh is a cell membrane impermeable intercalator and stains dead cells. In this experiment, cell viability was 92.1%. After the gating for the live cells, the singlets were selected from the 191Ir to 193 Ir bivariate plot. The long tail with low 191Ir to 193Ir signals corresponds to cell debris and fragments. Afterward, the singlet population is gated for CD20 and CD45 markers identified by 147Sm and 154Sm. For the population of singlet cells containing CD20 and CD45 markers, the 89Y signals from the NaYF4:Yb,Er NPs are plotted as a histogram. The Y-axis represents the number of cells, and the Xaxis represents the yttrium ion signal intensity detected per cell. Nanoparticles per Cell from Mass Cytometry Measurements. The average number of NPs associated per cell (XNP) can be calculated from the average metal signal xLn determined from the histogram of signal distribution in the FlowJo analysis using eq 2 XNP =

xln T × LnNP

(2)

Here, T is the transmission coefficient of the metal ion, and LnNP is the number of metal isotopes per NP.

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RESULTS AND DISCUSSION Encapsulation of individual NPs by liposomes is most effective for large NPs, since the limited curvature of the lipids makes it difficult to accommodate small NPs. There are examples in the literature of the encapsulation of gold48,49 and silica42,44 NPs with d ≈ 60 nm. This size would be too large for MC applications: a 60 nm NP contains ca. 1.3 × 106 Ln ions, and a high signal intensity of NPs will saturate the MC detector, which has a limit of 104 ion counts per mass channel per cell. For example, Ho has a transmission coefficient (T) of 2.4 × 4983

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the aqueous medium for the encapsulation, the nanoparticles aggregated during the sonication step. From a visual perspective, aggregation of the NPs led to obvious scattering of the green upconversion luminescence when the sample was excited with a 980 nm laser. In the absence of aggregation, as determined by DLS, we observed a uniform beam of upconversion luminescence. Results of DLS measurements of the NPs encapsulated with PEG-containing liposomes are presented in Figures 1D and 2.

Figure 1. Encapsulation of NaYF4:Yb,Er UCNPs in DEC221 liposomes. (A) Schematic of an empty DEC221 liposome, an UCNP, and the UCNP after encapsulation in the liposome. (B) TEM image of the citrate-coated NaYF4:Yb,Er NP sample (dTEM = 30 nm). (C) Coating protocol: thin films of the DEC221 lipid mixture and the mPEG-DSPE lipid were independently hydrated in water or HEPES buffer.48,49 They were mixed, combined with a dispersion of the UCNPs in water, and sonicated at 50 °C for 1 h. Then, the clear solution was passed 13 times through a 100 nm-pore filter to narrow the size distribution. (D) Comparison of CONTIN plots of DLS measurements on the citrate-coated NPs in water (red line, dh = 53 nm), empty DEC221 liposomes containing 6 mol % mPEG2K-DSPE prepared in HBS buffer (black line, dh = 65 nm), and an NP sample coated with DEC221 liposomes (containing 6 mol % mPEG2KDSPE) in HBS buffer (blue line, dh = 86 nm). Values of dh were calculated from a cumulant analysis of the autocorrelation decays.

Figure 2. DLS CONTIN plots of 30 nm UCNPs coated in water with DEC221 plus varying amounts (1, 2, 3, and 6 mol %) of PEG lipids. The NPs were purified by centrifugation at 15 000g for 30 min at 4 °C, followed by 13 passages through a 100 nm nanopore filter and by dilution.

The sample in Figure 1D containing 6 mol % mPEG2K-DSPE prepared in HBS buffer gave a size distribution that was symmetric and monomodal, with dh = 86 nm. In contrast, samples prepared in water, Figure 2, showed additional features in the CONTIN plots. The samples containing 2% and 3% mPEG2K-DSPE were clearly bimodal, whereas the samples prepared with 1% and 6% mPEG2K-DSPE exhibited a shoulder corresponding to more rapidly mobile species. The calculated dh value for the sample containing 6% mPEG2K-DSPE was 78 nm, not very different from that of the sample prepared in HEPES buffer. The position of the peak at smaller dh for the samples with 2% and 3% mPEG2K-DSPE is consistent with the presence of free UCNPs, not coated with lipid. As a consequence, we carried out further experiments to see if we could detect any free NPs. To obtain further information on the NP encapsulation, we examined the lipid-coated NP samples by TEM. Figure 3 shows the TEM images of two samples of NPs encapsulated with DEC221 + PEG2K-DSPE (6 mol %). Figure 3A,B shows NPs coated in water, and Figure 3C,D shows NPs coated in TBS buffer. There is no indication of NP clustering, and the magnified images in Figure 3B,D show a uniform gray corona approximately 5 to 6 nm thick around each of the NPs. Ip et al.49 also observed this type of gray corona with a similar thickness of around 60 nm Au NPs coated by the DEC221 lipid mixture and attributed the corona to the contrast associated with the lipid bilayer coating around individual NPs. Although we examined these TEM images carefully, we saw no indication of smaller species that might contribute to the shoulder at low d values in the plot for this sample in Figure 2. We did not see any free NPs without a gray corona characteristic of the lipid coating. We conclude that essentially all of the NPs are coated with lipid and the vast majority of the encapsulated NPs contain only a single NP.

Nanoparticle Encapsulation in PEG-Modified Liposomes. Liposomes for encapsulation of the 30 nm NaYF4:Yb,Er UCNPs were based on a ternary lipid mixture “DEC221”. This term refers to a 2:2:1 mol ratio of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (ESM), and cholesterol (Chol).48,49 As a reference, a thin film of DEC221 was hydrated in HEPES buffer at 50 °C to form a turbid suspension of multilamellar vesicles (MLVs). It was then sonicated at 50 °C until the solution turned clear (ca. 1 h), consistent with transforming the MLVs into unilamellar vesicles (ULVs).48,49 After 13 passages through a 100 nm-pore filter, a DLS measurement (Figure S4) showed a monomodal size distribution with dh = 43 nm. As described in the Experimental Section, this value was used to estimate the number of liposomes per 2 mg DEC221 sample. For NP encapsulation, we added varying amounts (1, 2, 3, 6 mol %, based on DEC221) of mPEG2K-DSPE to the lipid formulation.53 The PEG chains of mPEG2K-DSPE not only promote curvature of the coating but also impart “stealth” properties to minimize nonspecific interactions with cells. Figure 1 illustrates the protocol used to encapsulate the NPs in the lipid mixtures. Details are described in the Experimental Section. In a typical encapsulation, the yield of encapsulated NPs, as measured by ICP-MS after purification, was ca. 45%. NP encapsulation could be carried out effectively in HEPESbuffered saline (HBS) or in Tris-buffered saline (TBS) as well as in water. With either PBS or borate-buffered saline (BBS) as 4984

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Article

Chemistry of Materials

Figure 3. (A, B) TEM images of DEC221 coated UCNPs with 6 mol % mPEG2K-DSPE in water; (C, D) TEM images of DEC221 coated UCNPs with 6 mol % mPEG2K-DSPE in TRIS buffered saline. (E) Stability of NPs coated with DEC221 + mPEG2K-DSPE (6 mol %) in water and in PBS for 1 month in the presence of excess lipids. Error bars represent the standard deviation from three measurements. The dh values were obtained from the DLS measurements shown in Figure S5.

Figure 4. Mass cytometry histograms of Y signal obtained from DEC221-mPEG2K-DSPE coated NaYF4:Yb,Er UCNPs (with varying amounts (1, 2, 3, and 6 mol %) of PEGylated lipid) incubated for 30 min at 4 °C with 2 × 106 live Ramos cells. Dosages are 50 NPs/cell (1.7 fM NPs), 100 NPs/ cell (3.3 fM NPs), 1000 NPs/cell (33 fM NPs), and 10 000 NPs/cell (330 fM NPs). The counts in the inset tables refer to the mean intensity values obtained for yttrium from the mass cytometry measurements.

corresponding calculated values of dh and PDI) for measurements taken weekly over a 4 week period for a sample of 30 nm UCNPs coated in water with DEC221 + 6 mol % mPEG2KDSPE and then stored in water at 4 °C. After the sample came

While PBS buffer is problematic for the initial preparation of the lipid-coated NPs, the NPs coated and sonicated in water or HBS or TBS exhibit good colloidal stability after transfer to PBS. In Figure S5, we show a series of CONTIN plots (and the 4985

DOI: 10.1021/acs.chemmater.7b01339 Chem. Mater. 2017, 29, 4980−4990

Article

Chemistry of Materials

measured number of NPs per cell by the corresponding dosage. A summary of the calculated number of NPs/cell is presented in Figure 5.

to room temperature, aliquots were diluted into PBS or with water and examined by DLS. These plots all show a shoulder at low values of d, and some plots have a second peak at dh ≈ 20 nm. To summarize these results, we plot the z-averaged dh values for each sample in Figure 3E. One sees that the averaged values are about 20% larger for the coated UCNPs in PBS than for the same NPs in water. While both samples showed a small increase in hydrodynamic size over the first week, there was very little additional increase in particle size over 30 days, indicating good colloidal stability in PBS. Overall, the colloidal stability of UCNPs coated with DEC221 + mPEG2K-DSPE is superior to the oleate-capped UCNPs coated with a monolayer lipid coating.18 The bilayer lipid coating of NPs with DEC221 + mPEG2K-DSPE (6 mol %) provides long-term colloidal stability for the lanthanide NPs in PBS. This long-term colloidal stability is important for the use of these NPs as reporter tags in mass cytometry. Additionally, we would like to comment on the fact that the citrate-coated NPs in the presence of excess citrate ions showed reasonable colloidal stability in PBS buffer over a period of a few days, with no visible sign of flocculation. This observation is important for a control experiment with Ramos cells described below. When the citrate-coated NPs were washed to remove excess citrate, they rapidly flocculated in PBS buffer. Assessing Nonspecific Binding (NSB) to Cells by Mass Cytometry. In this section, we examine the interactions of liposome-encapsulated 30 nm UCNPs with cell suspensions. The liposome coating consisted of DEC221 plus various amounts of mPEG2K-DSPE. In these experiments, aliquots of 2 × 106 cells were transferred from growth medium to PBS buffer (100 μL) containing 0.5 wt % bovine serum albumin (BSA). The cells were treated with a cocktail of metal-tagged antibodies for 30 min at room temperature, followed by washing and resuspension in PBS (100 μL). The cells were then incubated for 30 min at 4 °C with different concentrations of NPs ranging from 50 NPs/cell (1.7 femtomolar (fM) NPs) to 30 000 NPs/cell (1.0 nanomolar (nM) NPs). Low temperature incubation was used to minimize active uptake (e.g., endocytosis) of the NPs by the cells. After washing the cells to remove free NPs, the cells were fixed and treated with an Ir-containing DNA intercalator. The cells were examined by mass cytometry, and the data was analyzed with FlowJo software. Cell events were identified by the 191Ir and 193Ir signals from the intercalator as well as by isotopes carried by metal-conjugated antibodies specific to biomarkers on the cell surface. Ramos Cells. For initial experiments, we chose the Ramos cell line (B-cell lymphoma) to examine the extent of NP nonspecific binding. The cells were stained with CD45-147Sm and CD20-154Sm. The gating protocol for identifying cell events and 89Y signals originating from the NPs is shown in Figure S6. A real time instrument viewer snapshot showing the various isotopes detected for individual cell events is presented in Figure S7. Since we are seeking to minimize NSB, low 89Y signals are desirable. Figure 4 presents histograms obtained from the FlowJo software for the 89Y signal distribution. In these plots, the Y-axis represents the number of cells and the Xaxis represents the 89Y signal per cell. Using the average yttrium signal intensity per cell from the histogram, one can calculate the number of NPs per cell. This calculation employs the transmission coefficient of yttrium (3.63 × 10−5) and the number of yttrium atoms (1.4 × 105) per 30 nm NaYF4:Yb,Er UCNP. The percent of NSB can be calculated by dividing the

Figure 5. Bar graph indicating the level of NSB, expressed as the calculated number of bound NPs per cell from the nonspecific binding assay of DEC 221 coated NaYF4:Yb,Er (d = 30 nm) with Ramos cells for coatings containing different amounts of mPEG-DSPE. Different dosages ranging from 50 NPs/cell (1.7 fM NPs) to 100 NPs/cell (3.3 fM NPs), 1000 NPs/cell (33 fM NPs), and 10 000 NPs/cell (330 fM NPs) were incubated with 2 × 106 Ramos cells at 4 °C for 30 min. The NP number was converted from the Y count (Figure 4) using the transmission coefficient of Y and the number of Y atoms per NP. The error bars represent the standard deviation of the mean intensity. The table (inset) shows the calculated number of NPs for each experiment.

We begin by examining the data in Figure 4. In MC, the background signal is very low, on the order of one count during the transit time of the ion cloud generated by an individual cell through the instrument. Thus, one considers a mean value of 1 to be background. Taking this idea into account, we can conclude that, at dosages of 50 and 100 NPs/cell (Figures 4 and 5), MC detects no nonspecific binding to cells for liposome-encapsulated NPs containing the various percentages of mPEG-DSPE. In case of 1000 NPs/cell, we observe some NSB for all four levels of mPEG-DSPE content of the liposomes (Figure 4C). When we convert the counts observed for each mPEG-DSPE dosage to the number of NPs/cell (Figure 5), the NSB is ca. 0.5−2 NPs/cell, less than 0.2% of the NP dosage. In Figure 4D, we observe that the NSB has increased substantially at 10 000 NPs/cell. The sample coated with DEC221 + 6 mol % mPEG-DSPE shows the highest amount of NSB, whereas the sample containing 3 mol % mPEG-DSPE shows the lowest amount. However, if we convert the signal intensity of 89Y to NPs/cell and take into account the standard deviation in the values (Figure 5), we see that the percentage of nonspecific binding for these two samples is not very different. Overall, we find that the extent of NSB compared to dosage is very small (