Surface modification of NaLnF4 nanoparticles with bisphosphonic acid derivatives for mass cytometry applications

Abstract

Described herein are nanoparticles comprising NaLnF4 (wherein Ln includes all non-radioactive lanthanide elements) or NaYF4, and methods of making and using the same. The nanoparticles may be modified with PEG or zwitterionic polymers containing bisphosphonic acid end groups, and the bisphosphonic acid end groups may include aminohexyl groups. As described herein, NaLnF4 or NaYF4 nanoparticles may be used as highly sensitive reagents for mass cytometry.

Description

[Technical field]

[0001]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 352,615, filed June 15, 2022, and U.S. Provisional Patent Application No. 63 / 315,524, filed March 1, 2022, the entire contents of both of which are incorporated by reference herein in their entirety for all purposes. [Background technology]

[0002]

[0002] Mass cytometry (MC) is a high-throughput multiparameter bioanalytical technique for single cell analysis. Imaging mass cytometry (IMC) is an extension of MC that allows the measurement of cell markers on tissue sections by combining a laser ablation system with a mass cytometer. Reagents for MC and IMC™ include Ab-conjugated metal chelating polymers that carry approximately 150 metal ions. To increase sensitivity, reagents with a high number of metal atoms per mass tag are desirable. Summary of the Invention

[0003] Mass cytometry (MC) is a high-throughput multiparameter bioanalytical technique for single cell analysis. In this technique, cell suspensions are labeled with antibodies (Abs) tagged with heavy metal isotopes. The cells are individually but stochastically injected into an inductively coupled plasma time-of-flight mass spectrometer, and the signal at the detector is directly proportional to the number of ions per Ab-conjugate. MC is described in PCT Application No. PCT / US2021 / 049667, the contents of which are incorporated herein by reference for all purposes. MC instruments cover a mass range of 89(Y)-220(Bi) and can analyze up to 100 parameters per cell. Imaging mass cytometry (IMC) is an extended form of MC that combines a laser ablation system with a mass cytometer to allow for the measurement of cell markers on tissue sections. Reagents for MC and IMC™ include Ab-conjugated metal chelating polymers carrying approximately 150 metal ions. To increase sensitivity, reagents with a high number of metal atoms per mass tag are desirable. As described herein, lanthanide nanoparticles (LnNPs) in the form of NaLnF4 have a mass tag density of approximately 10 LnNPs with diameters of 10-20 nm. 5 Since it contains 1 Ln ions, it is a good candidate for this purpose. In some examples, the NaLnF4 nanoparticles (NPs) described herein can be used for biological applications such as in vitro cell labeling, in vivo imaging, and cell tracking.

[0004]

[0004] As described herein, NaLnF4 or NaYF4 NPs can be used as highly sensitive reagents for mass cytometry. These NPs can be uniform in size, colloidally stable in phosphate buffer (i.e., PBS) and biological media, and equipped with functionalities for attaching antibodies to avoid non-specific interactions with cells or tissue samples. Prevention of non-specific binding has been a significant challenge. One approach to minimize or reduce non-specific binding is to modify the NP surface with polyethylene glycol (PEG). On the other hand, an alternative to PEG is surface modification with zwitterionic polymers. The zwitterionic moiety contains a positively charged group and a negatively charged group linked by a short carbon chain. Without being bound by theory, zwitterionic polymers appear to have reduced biological interactions compared to PEG, which may be due to the superhydrophilic nature of zwitterions.

[0005]

[0005] Provided herein are nanoparticles comprising NaLnF4 (wherein Ln includes all non-radioactive lanthanide elements) or NaYF4. The nanoparticles described herein may be modified with PEG or zwitterionic polymers containing bisphosphonic acid end groups, which may include aminohexyl groups.

[0006]

[0006] Also provided herein is a method of analyzing a cell, comprising introducing the cell into a nanoparticle described herein.

[0007] Also provided herein is a mass cytometry system comprising any one of the nanoparticles described herein.

[0008]

[0008] Also provided herein is a mixture of nanoparticles, the mixture comprising a plurality of nanoparticles, each nanoparticle being any one of the nanoparticles described herein.

[0009]

[0009] While not wishing to be bound by any particular theory, the present specification may discuss underlying beliefs or understandings related to embodiments of the present invention. It is recognized that regardless of whether a mechanistic explanation or hypothesis is ultimately correct, embodiments of the present invention may nevertheless be operable and useful. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 depicts a nanoparticle core with a corona according to an embodiment of the present invention. [Figure 2A] FIG. 13 illustrates the transfer of oleic acid coated nanoparticles into aqueous media using citrate exchange according to an embodiment of the present invention. [Figure 2B] FIG. 1 depicts a narrow size distribution of citrate coated nanoparticles according to an embodiment of the present invention. [Figure 2C] FIG. 2 depicts a transmission electron microscope (TEM) image of citric acid coated nanoparticles according to an embodiment of the present invention. [Diagram 3] FIG. 1 depicts the synthesis and ligand exchange of PEG-neridronic acid according to an embodiment of the present invention. [Figure 4A] FIG. 1 depicts a graph of intensity versus diameter for NPs in water according to an embodiment of the present invention. [Figure 4B] FIG. 1 depicts a graph of intensity versus diameter for mPEG2000-Ner coated NPs in PBS according to an embodiment of the present invention. [Figure 4C] FIG. 1 depicts a TEM image of mPEG2000-Ner coated NPs according to an embodiment of the present invention. [Figure 5A] FIG. 13 depicts the signal in the 89Y channel of the sample in 3% BSA prepared in Example 4 according to an embodiment of the present invention. [Figure 5B] FIG. 13 shows the signal in the 174Yb channel of the sample in 3% BSA prepared in Example 4 according to an embodiment of the present invention. [Figure 6A] FIG. 1 depicts the synthesis of zwitterionic polymer PSBMA-Ner according to an embodiment of the present invention. [Figure 6B] FIG. 1 depicts three different nanoparticles tested, namely, NPs incubated with 100% PSBMA-Ner, NPs incubated with 50% mPEG-Ner and 50% PSBMA-Ner, and NPs incubated with 100% mPEG-Ner, according to embodiments of the present invention. [Figure 7A] FIG. 1 depicts the characterization of NPs with 50% mPEG-Ner and 50% PSBMA-Ner according to an embodiment of the present invention. [Figure 7B] FIG. 1 depicts characterization of NPs with 100% PSBMA-Ner according to an embodiment of the present invention. [Figure 8A] FIG. 13 depicts the results of non-specific binding of NPs according to an embodiment of the present invention. [Figure 8B] FIG. 13 depicts the results of non-specific binding of NPs according to an embodiment of the present invention. [Figure 9] 1 is a table depicting the surface polymer composition of NPs according to embodiments of the present invention. [Figure 10] 1 shows NPs of different lengths tested according to embodiments of the present invention. The NPs tested include NPs with 100% mPEG2000-Ner, NPs with about 50% mPEG2000-Ner and about 50% PSBMA-Ner, NPs with about 50% N3-PEG2000-Ner and about 50% PSBMA-Ner, NPs with about 50% mPEG5000-Ner and about 50% PSBMA-Ner, and NPs with about 50% N3-PEG5000-Ner and about 50% PSBMA-Ner. N3 is an azide functional group. [Figure 11] FIG. 13 depicts non-specific binding results from suspension mass cytometry experiments of NPs according to an embodiment of the present invention. [Figure 12] FIG. 1 depicts the conjugation of an antibody (CD45RO or PD-1) onto NPs with about 50% N3-PEG5000-Ner and about 50% PSBMA-Ner using DBCO-azide click chemistry according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011]

[0029] Mass cytometry (MC) is a high-throughput multiparameter bioanalytical technique for single-cell analysis. In this technique, cell suspensions are labeled with antibodies (Abs) tagged with heavy metal isotopes. Cells are individually but stochastically injected into an inductively coupled plasma time-of-flight mass spectrometer, and the signal at the detector is directly proportional to the number of ions per Ab-conjugate. MC is described in PCT Application No. PCT / US2021 / 049667, the entire contents of which are incorporated herein by reference for all purposes. MC instruments cover a mass range of 89(Y)-220(Bi) and can analyze up to 100 parameters per cell. Imaging mass cytometry (IMC) is an extended form of MC that combines a laser ablation system with a mass cytometer to allow for the measurement of cellular markers on tissue sections. Reagents for MC and IMC™ include Ab-conjugated metal chelating polymers carrying approximately 150 metal ions. To increase sensitivity, reagents with a high number of metal atoms per mass tag are desirable. Lanthanide nanoparticles (LnNPs) in the form of NaLnF4 as described herein have a mass tag density of approximately 10 LnNPs with diameters of 10-20 nm. 5 Since it contains 1 Ln ions, it is a good candidate for this purpose. In some examples, the NaLnF4 nanoparticles (NPs) described herein can be used for biological applications such as in vitro cell labeling, in vivo imaging, and cell tracking.

[0012]

[0030] NaLnF4 or NaYF4 NPs can be used as sensitive reagents for mass cytometry. These NPs can be uniform in size, colloidally stable in phosphate buffer (i.e., PBS) and biological media, and equipped with functionalities for attaching antibodies to avoid non-specific interactions with cells or tissue samples. Prevention of non-specific binding has been a significant challenge. One approach to minimize or reduce non-specific binding is to modify the NP surface with polyethylene glycol (PEG). On the other hand, an alternative to PEG is surface modification with zwitterionic polymers. The zwitterionic moiety contains positively and negatively charged groups linked by a short carbon chain. Without being bound by theory, zwitterionic polymers appear to have reduced biological interactions compared to PEG, which may be due to the superhydrophilicity of zwitterions.

[0013]

[0031] Some embodiments described herein use uniform NaYF4:Yb / Er NPs (approximately 25 nm in diameter) modified with PEG or zwitterionic polymers containing bisphosphonic acid end groups.

[0014]

[0032] As described herein and shown in FIG. 1, the nanoparticle core may include a corona, which may be attached to an antibody. In some embodiments, the core has a narrow size distribution (e.g., coefficient of variation [CV] <5%). Without being bound by theory, the narrow size distribution of the core helps to keep the number of ions per mass tag constant. Without being bound by theory, the corona promotes colloidal stability in phosphate buffer. In some examples, the corona may suppress non-specific binding to cells. In some examples, the corona may also have functionality for antibody conjugation. The diameter of the nanoparticle core described herein may be about 10 nm to about 50 nm, which may be measured from transmission electron microscope (TEM) images. The overall hydrodynamic diameter (d h ) can be measured, for example, by dynamic light scattering.

[0015]

[0033] Neridronic acid (Ner) is an aminohexyl bisphosphonic acid. As described herein, in some examples, PEG is conjugated to Ner in a 1:1 ratio using the amine functional group of Ner (PEG-Ner). In another example, instead of PEG, a zwitterionic polymer, poly(sulfobetaine methacrylate) (PSBMA), was prepared by RAFT polymerization and Ner was conjugated to one end of the PSBMA (PSBMA-Ner). In some examples, nanoparticles (NPs) with coatings including PEG-Ner, PSBMA-Ner, and / or PEG-Ner / PSBMA-Ner mixtures by ligand exchange are also described herein. In some examples, the modified NPs were characterized by examining their morphology, colloidal stability, surface polymer composition, and nonspecific binding to tissue samples. In certain non-limiting examples, no change in morphology was observed after ligand exchange with either PEG-Ner or PSBMA-Ner. In some examples, NPs coated with PEG-Ner, PSBMA-Ner and / or PEG-Ner / PSBMA-Ner showed long-term colloidal stability in PBS (e.g., were colloidally stable for at least 24 hours, 7 days, 28 days or 64 days). In some embodiments, IMC™ testing showed that the PEG-Ner coated NPs described herein exhibited a significant amount of non-specific binding, while the PSBMA-Ner modified NPs described herein significantly reduced non-specific binding, even when used as a mixture with PEG-Ner. In some examples described herein, functional PEG can introduce Abs for target binding studies, along with PSBMA to reduce non-specific binding.

[0016]

[0034] 1. Nanoparticles containing NaLnF4 Described herein are nanoparticles comprising MLnF4 or MYF4, where M is Li, Na, Rb or Cs, and Ln includes all non-radioactive lanthanide elements. Also described herein are nanoparticles comprising NaLnF4 (where Ln includes all non-radioactive lanthanide elements) or NaYF4. In some embodiments, Ln includes La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, or combinations thereof.

[0017]

[0035] In some embodiments, the nanoparticles further comprise a dopant, hi some embodiments, the dopant comprises Yb or Er.

[0018]

[0036] In some embodiments, the nanoparticles are modified with PEG or zwitterionic polymers containing bisphosphonic acid end groups.

[0019]

[0037] In some embodiments, the nanoparticles are modified with zwitterionic polymers containing bisphosphonic acid end groups, where the bisphosphonic acid end groups comprise aminohexyl bisphosphonic acid. In some embodiments, the aminohexyl bisphosphonic acid comprises neridronic acid. In some embodiments, the aminohexyl bisphosphonic acid comprises alendronic acid. In some embodiments, the alendronic acid is conjugated to one end of poly(sulfobetaine methacrylate) (PSBMA). In some embodiments, the alendronic acid is conjugated to one end of PEG.

[0020]

[0038] In some embodiments, the nanoparticles are modified with PEG or zwitterionic polymers containing bisphosphonic acid end groups, and the nanoparticles are modified with poly(sulfobetaine methacrylate) (PSBMA). In some embodiments, the nanoparticles are modified with zwitterionic polymers, and the bisphosphonic acid end groups of the zwitterionic polymer comprise aminohexyl bisphosphonic acid, and the nanoparticles are modified with poly(sulfobetaine methacrylate) (PSBMA).

[0021]

[0039] In some embodiments, the nanoparticles are modified with a zwitterionic polymer, and the bisphosphonic acid end group of the zwitterionic polymer comprises an aminohexyl bisphosphonic acid, including neridronic acid, where the nanoparticles are modified with poly(sulfobetaine methacrylate) (PSBMA). In some embodiments, neridronic acid is conjugated to one end, and the neridronic acid is conjugated to PSBMA (PSBMA-Ner). In some embodiments, the nanoparticles are further modified with PEG. In some embodiments, the PEG is PEG 2000 or PEG 5000 In some embodiments, the azide functional group is conjugated to PEG. In another embodiment, the PEG is conjugated to neridronic acid (PEG-Ner).

[0022]

[0040] In some embodiments, the nanoparticles described herein are modified with PSBMA conjugated at one end to neridronic acid (PSBMA-Ner) and PEG conjugated at one end to neridronic acid (PEG-Ner). In some embodiments, the PSBMA-Ner represents 30%-40%, 40%-50%, or 50%-60% of the total of the PSBMA-Ner and PEG-Ner modifications.

[0023]

[0041] In some embodiments, the nanoparticles are modified with a zwitterionic polymer, and the bisphosphonic acid end group of the zwitterionic polymer comprises an aminohexyl bisphosphonic acid, including alendronic acid, where the nanoparticles are modified with poly(sulfobetaine methacrylate) (PSBMA). In some embodiments, the alendronic acid is conjugated to one end, and the alendronic acid is conjugated to PSBMA (PSBMA-alendronic acid). In some embodiments, the nanoparticles are further modified with PEG. In some embodiments, the PEG is PEG 2000 or PEG 5000 In some embodiments, the azide functional group is conjugated to PEG. In another embodiment, the PEG is conjugated to alendronate (PEG-alendronate).

[0024]

[0042] In some embodiments, the nanoparticles described herein are modified with PSBMA conjugated to alendronate at one end and PEG conjugated to alendronate at one end (PEG-alendronate). In some embodiments, the PSBMA-alendronate is 30%-40%, 40%-50%, or 50%-60% of the sum of the PSBMA-alendronate and PEG-alendronate modifications.

[0025]

[0043] In some embodiments, the nanoparticles described herein can have a diameter in the range of 10 nm to 50 nm (e.g., 15 nm to 45 nm, 20 nm to 40 nm, or 25 nm to 35 nm). In some instances, the diameter is measured by transmission electron microscopy.

[0026]

[0044] In some embodiments, the nanoparticles described herein have a molecular weight of at least 10 5 Ln ions (e.g., at least 10 3 , 10 4 , 10 5 , 10 6 , 107 or 10 8 Ln ions).

[0027]

[0045] The nanoparticles described herein can be attached to one or more antibodies or one or more other biocompatible agents. In some embodiments, the nanoparticles include antibodies. In some embodiments, the nanoparticles include other biocompatible agents. In some embodiments, the nanoparticles include at least one or more antibodies and at least one or more other biocompatible agents. In some embodiments, the antibodies include, but are not limited to, CD45RO or PD-1. In some embodiments, the antibodies include IgG or IgM from mouse, rabbit, rat, goat, hamster, or similar species. In some embodiments, the antibodies include antibodies suitable for use in flow cytometry, immunohistochemistry, or immunofluorescence. In some embodiments, the other biocompatible agents include, but are not limited to, oligonucleotides, avidin-type proteins, aptamers, lectins, or other biomolecules that specifically bind to target biomolecules.

[0028]

[0046] 2. How to Analyze Cells Also described herein is a method of analyzing cells, comprising introducing any one of the nanoparticles described herein into the cells. In some examples, the method further comprises analyzing a mixture of cells and one or more of the nanoparticles described herein. In some examples, the analyzing step comprises analyzing the mixture of cells and nanoparticles by mass cytometry. In some examples, the method comprises delivering the mixture of cells and nanoparticles to a mass cytometry system comprising an inductively coupled plasma source and an analysis device. In some examples, the method further comprises an ignition step in which the mixture of cells and nanoparticles is ignited by the inductively coupled plasma source. In some examples, the method further comprises detecting the mixture of cells and nanoparticles with the analysis device.

[0029]

[0047] 3. Mass Cytometry System Also described herein is a mass cytometry system comprising any one of the nanoparticles described herein. In some examples, the mass cytometry system comprises one or more of the nanoparticles described herein. The mass cytometry system can include an inductively coupled plasma source and an analyzer. The analyzer can include a mass spectrometer or an imaging mass spectrometer.

[0030]

[0048] 4. Nanoparticle Mixture Also described herein are mixtures of nanoparticles comprising a plurality of nanoparticles, each nanoparticle being any one of the nanoparticles described herein, in some embodiments, the plurality of nanoparticles comprises at least 10, 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 nanoparticles.

[0031]

[0049] The present invention may be further understood by the following non-limiting examples. EXAMPLES

[0032]

[0050] PSBMA was synthesized and neridronic acid was attached to its termini. Samples of NaYF4:Yb / Er NPs were coated with PEG-neridronic acid, PSBMA-neridronic acid, and / or a mixture of PEG-neridronic acid and PSBMA-neridronic acid. Low nonspecific binding was desired because neither PEG-neridronic acid nor PSBMA-neridronic acid had a targeting agent on the polymer. In these experiments, PEG-neridronic acid showed significant nonspecific binding, whereas PSBMA-neridronic acid showed very low levels of nonspecific binding. NaYF4:Yb / Er NPs coated with a mixture of PEG-neridronic acid and PSBMA-neridronic acid also showed very low levels of nonspecific binding. By including PEG-neridronic acid in the mixture, it becomes possible to attach antibodies or other biocompatible agents to the NPs using the heterobifunctional PEG-neridronic acid in these mixed coatings.

[0033]

[0051] Similar materials may include alendronic acid in place of neridronic acid, for example any nanoparticle with neridronic acid may have alendronic acid in place of neridronic acid.

[0034]

[0052] Example 1: The oleic acid coated nanoparticles described herein were transferred to aqueous media using citrate exchange, as shown in Figure 2 A. Without being bound by theory, citrate exchange is rapid, safe, and may allow for long-term storage of the nanoparticles.

[0035]

[0053] As shown in Figure 2B, a narrow size distribution of the citrate-coated nanoparticles was observed. The z-average hydrodynamic diameter (d Z ) was 31.6 nm. The polydispersity index (PDI) was 0.03.

[0036]

[0054] As shown in Figure 2C, a transmission electron microscope (TEM) image of the citric acid-coated nanoparticles was obtained, and the average diameter of the nanoparticles in the TEM (d TEM ) was 28 nm. No change in the morphology of the NPs was observed after citrate exchange.

[0037]

[0055] Example 2: In one embodiment, the synthesis and ligand exchange of PEG-neridronic acid described herein is shown in Figure 3. As shown in Figure 3, mPEG 2000 -NHS combined with neridronic acid and mPEG 2000 mPEG-Ner was formed. 2000 Nanoparticles as described herein coated with mPEG were prepared by a ligand exchange technique with citrate. Thus, a solution of nanoparticles as described herein coated with citrate and a citrate buffer solution was prepared. Excess citrate in the solution was removed by spin filtration. Next, mPEG was added to the nanoparticles. 2000 -NER was added to the solution and the solution was incubated at room temperature for 24 hours. 2000 The mPEG-NER was removed by spin filtration with water or, optionally, by spin filtration with a PBS solution. 2000 -Ner coated nanoparticles were thus obtained in water or PBS.

[0038]

[0056] Example 3: Some examples of nanoparticles described herein were characterized by dynamic light scattering (DLS) and TEM. Figure 4A shows a graph of intensity versus diameter for NPs in water. The citrate-coated NPs shown in the graph of Figure 2B are shown in the left trace in Figure 4A. mPEG 2000 The mPEG-Ner coated NPs were observed to have an average diameter of 37.9 nm and a PDI of 0.04. 2000 The average diameter of -Ner-coated NPs was observed to increase by approximately 6.3 nm compared to citrate-coated NPs, as can be seen in Figure 4A.

[0039]

[0057] FIG. 4B shows mPEG in PBS. 2000 1 shows a graph of intensity versus diameter for mPEG-Ner coated NPs. The average diameter was observed to be 39.4 nm. The PDI was observed to be 0.03. 2000The -Ner-coated NPs were observed to be colloidally stable in PBS for at least 28 days.

[0040]

[0058] Figure 4C shows mPEG 2000 TEM images of mPEG-Ner coated NPs. 2000 No changes in the morphology of NPs were observed after coating with -Ner.

[0041]

[0059] Example 4: Using tissue samples prepared by the immunohistochemistry protocol, mPEG 2000 The non-specific binding of -Ner-coated NaYF4:Yb / Er NPs was tested. Normal human tonsil tissue was subjected to deparaffinization, hydration and antigen retrieval. The tissue sample was then subjected to blocking with BSA buffer. The tissue sample was then stained with a metal-tagged antibody cocktail containing antibodies and NPs (without antibodies). The stained tissue sample was analyzed on an IMC™ from Standard BioTools Inc.

[0042]

[0060] Example 5: There should be no specific binding since there was no antibody on the NP in Example 4. Any signal in the imaging mass cytometry is the result of non-specific binding.

[0043]

[0061] The NP concentration on the tissue was 2 × 10 10 The concentration was NP / mL. Intensity below 5 is background. Intensity above 5 is signal.

[0044]

[0062] FIG. 5A shows the results of the analysis of the samples in 3% BSA prepared in Example 4. 89 The signal in the Y channel is shown. The average intensity was 180.9. Figure 5A shows the non-specific binding.

[0045]

[0063] FIG. 5B shows the results of the analysis of the samples in 3% BSA prepared in Example 4. 174 The signal in the Yb channel is shown. The average intensity was 174.7. Figure 5B shows the non-specific binding.

[0046]

[0064] Example 6: A zwitterionic polymer was added in an attempt to reduce non-specific binding. Figure 6A depicts the synthesis of the zwitterionic polymer PSBMA-Ner.

[0047]

[0065] Figure 6B represents the three different nanoparticles tested, namely, NPs incubated with 100% PSBMA-Ner, NPs incubated with 50% mPEG-Ner and 50% PSBMA-Ner, and NPs incubated with 100% mPEG-Ner.

[0048]

[0066] Example 7: Figure 7A shows the characterization of NPs with 50% mPEG-Ner and 50% PSBMA-Ner. The average diameter of these NPs was observed to be 42.7 nm. The PDI was 0.04.

[0049]

[0067] Figure 7B shows the characterization of NPs with 100% PSBMA-Ner. The average diameter was observed to be 43.7 nm. The PDI was 0.05.

[0050]

[0068] Both samples were colloidally stable in PBS and had narrow size distributions by DLS. No changes in morphology were observed by TEM.

[0051]

[0069] Example 8: Figures 8A and 8B show the results of non-specific binding of NPs described in Figure 6B. Figure 8A shows that NPs with 100% mPEG-Ner still show a signal (intensity 180.9) indicative of non-specific binding. Figure 8B shows that NPs with 50% mPEG-Ner and 50% PSBMA-Ner and NPs with 100% PSBMA-Ner show signals consistent with the background (intensity 4.2 for 50% / 50% and intensity 2.9 for 100% PSBMA-Ner). Figure 8B shows low non-specific binding when NPs are coated with PSBMA-Ner. The addition of zwitterionic polymer was observed to significantly reduce non-specific binding, so that no signal was observed.

[0052]

[0070] Example 9: 9 is a table showing the surface polymer composition of NPs coated with the polymers described herein. 2000 -NER (1 mmol / L (mM)), (2) mPEG 2000 The surface composition of the NPs, as described in Figure 9, was determined by incubating the NPs with either PSBMA-Ner (0.5 mM) or PSBMA-Ner (0.5 mM), or (3) PSBMA-Ner (1 mM). 31 P content and PSBMA-Ner 32 The S content was quantified by ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy) measurement. For all three compositions, the mPEG per NP 2000 The number of PSBMA-Ner and PSBMA-Ner per NP was obtained and the results are shown in FIG.

[0053]

[0071] Example 10: PEGs of different lengths are examined to see how they affect nonspecific binding. PEGs with average molecular weights of 2,000 and 5,000 were examined. The higher molecular weight PEGs are longer than the lower molecular weight PEGs.

[0054]

[0072] Figure 10 shows the NPs tested for different lengths. The NPs tested included 100% mPEG2000 -Ner-containing NPs, approximately 50% mPEG 2000 -Ner and about 50% PSBMA-Ner, and about 50% N3-PEG 2000 NPs with about 50% mPEG-Ner and about 50% PSBMA-Ner 5000 -Ner and about 50% PSBMA-Ner, and NPs with about 50% N3-PEG 5000 The NPs contain -Ner and about 50% PSBMA-Ner. N3 is an azide functional group.

[0055]

[0073] Example 11: FIG. 11 shows the non-specific binding results from the suspension mass cytometry experiment of NPs described in FIG. 10. The x-axis shows the signal intensity. The y-axis is the number of cells counted at each signal intensity. PEG 5000 and PSBMA-Ner-bearing NPs were PEG 2000 As shown in Figure 11, a metal chelating polymer (Maxpar®) is shown as a control with little to no nonspecific binding.

[0056]

[0074] Example 12: The antibody was conjugated to the NP. Figure 12 shows that approximately 50% N3-PEG 5000 Figure 1 shows the conjugation of antibodies (CD45RO or PD-1) onto NPs with -Ner and approximately 50% PSBMA-Ner using DBCO-azide click chemistry.

[0057]

[0075] INCORPORATION BY REFERENCE AND VARIATION STATEMENT Throughout this application, all references, e.g., patent documents, including issued or granted patents or equivalents and patent application publications, as well as non-patent documents or other materials, are incorporated herein by reference in their entirety, as if each was individually incorporated by reference. No reference is admitted to be prior art.

[0058]

[0076] When groups of substituents are disclosed herein, it is understood that each and every individual member of the group, as well as any and all subgroups and classes that can be formed using those substituents, are disclosed separately. When Markush groups or other groupings are used herein, each and every individual member of the group, as well as any and all possible combinations and subcombinations of the group, are intended to be included separately in the disclosure. As used herein, "and / or" means that one, all, or any combination of the items in the list separated by "and / or" is included in the list, for example, "1, 2, and / or 3" is equivalent to "1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2, and 3."

[0059]

[0077] Unless otherwise stated, the invention can be practiced using any formulation or combination of the components described or exemplified. The specific names of the materials are for illustrative purposes, as it is recognized that one skilled in the art may name the same materials differently. It will be understood that methods, device elements, starting materials, and synthetic methods other than those specifically exemplified can also be used in the practice of the invention without resorting to undue experimentation. For any such methods, device elements, starting materials, and synthetic methods, all functional equivalents known in the art are intended to be encompassed by the present invention. Whenever a range is given herein, such as a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values ​​contained within the given range, are intended to be encompassed by the present disclosure. Each intervening value between the upper and lower limits of the range, to one tenth of the unit of the lower limit, is also understood to be specifically disclosed, unless the context clearly requires otherwise. Each smaller range between any stated or intervening value within a stated range and any other stated or intervening value within that stated range is also encompassed. The upper and lower limits of these smaller ranges may independently be included or excluded from the ranges, and ranges including either limit, neither limit, or both limits are each encompassed within the invention, provided that any specifically excluded limit in a stated range is subject to the invention. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0060]

[0078] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended, and does not exclude additional unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any reference herein to the term "comprising," particularly in a description of a component of a composition, a description of a method, or a description of an element of a device, is understood to encompass a composition, method, or device that consists essentially of, and consists of, the recited components or elements, optionally in addition to other components or elements. The inventions illustratively described herein may suitably be practiced in the absence of any one or more elements or one or more limitations not specifically disclosed herein.

[0061]

[0079] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly requires otherwise. Thus, for example, a reference to "a method" includes a plurality of such methods, a reference to "the nanoparticle" includes a reference to one or more nanoparticles and equivalents thereof known to those skilled in the art, and so forth. The terms and expressions used are used in a descriptive and not limiting sense, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described herein or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that those skilled in the art may resort to modifications and variations of the concepts disclosed herein, and such modifications and variations are deemed to be within the scope of the invention as defined by the appended claims.

Claims

1. NaLnF ４ where Ln includes all non-radioactive lanthanide elements, or NềYF ４ The nanoparticles include

2. The nanoparticle of claim 1 further comprising a dopant.

3. The nanoparticle of claim 2 , wherein the dopants include Yb and Er.

4. The nanoparticles according to any one of claims 1 to 3, which are modified with PEG or zwitterionic polymers containing bisphosphonic acid end groups.

5. 5. The nanoparticle of claim 4, wherein the nanoparticle is modified with the zwitterionic polymer and the bisphosphonic acid end group comprises aminohexyl bisphosphonic acid.

6. The nanoparticle of claim 5 , wherein the aminohexylbisphosphonic acid comprises neridronic acid.

7. The nanoparticle of claim 5 , wherein the aminohexyl bisphosphonic acid comprises alendronic acid.

8. The nanoparticle of claim 7, wherein the alendronic acid is conjugated to one end of poly(sulfobetaine methacrylate) (PSBMA).

9. The nanoparticle of claim 8, further modified with alendronic acid conjugated to one end of the PEG.

10. The nanoparticles according to any one of claims 4 to 6, which are modified with poly(sulfobetaine methacrylate) (PSBMA).

11. The nanoparticle of claim 10, wherein the neridronic acid is conjugated to one end (PSBMA-Ner).

12. The nanoparticle of claim 10 or 11, further modified with PEG.

13. The PEG is ２０００ or PEG ５０００ The nanoparticle of claim 12, wherein

14. The nanoparticle of claim 12 or 13, wherein an azide functional group is conjugated to the PEG.

15. The nanoparticles according to claim 12 or 13, wherein the PEG is conjugated to neridronic acid (PEG-Ner).

16. 2. The nanoparticle of claim 1, which is modified with PSBMA conjugated at one end to neridronic acid (PSBMA-Ner) and PEG conjugated at one end to neridronic acid (PEG-Ner).

17. The nanoparticle of claim 16, wherein the PSBMA-Ner is 30% to 40%, 40% to 50%, or 50% to 60% of the sum of the PSBMA-Ner and PEG-Ner modifications.

18. The nanoparticles according to any one of claims 1 to 17, wherein the diameter of the nanoparticles, as measured by transmission electron microscopy, is in the range of 10 nm to 50 nm.

19. At least 10 ５ The nanoparticles according to any one of claims 1 to 18, comprising Ln ions.

20. The nanoparticle of any one of claims 1 to 19, attached to an antibody or other biocompatible agent.

21. A method for analysing a cell, comprising introducing said cell into a nanoparticle according to any one of claims 1 to 20.

22. A mass cytometry system comprising the nanoparticles of any one of claims 1 to 19.

23. A mixture of nanoparticles comprising a plurality of nanoparticles, each nanoparticle being a nanoparticle according to any one of claims 1 to 19.

24. 24. The mixture of claim 23, wherein the plurality of nanoparticles comprises at least 10, 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 nanoparticles.

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