STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant No. FD005266 awarded by the Food and Drug Administration. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62/624,373, fded 31 January 2018 and entitled“A Comprehensive Method to Quantify Iron Distribution in Plasma” in the name of Sarah L.J. Michel et al., incorporated herein by reference in its entirety.

FIELD

[0003] The present invention relates to methods of quantifying metal species present in a sample after a metal-containing nanoparticle, e.g., an iron-containing nanoparticle drug, has been administered to a subject, said method using Liquid Chromatography -Inductively Coupled Plasma-Mass Spectrometry (LC-ICP-MS). This method has potential applications not only for quantifying iron species in vivo after the administration of iron-containing nanoparticle drugs but also for quantifying other metal species in vivo following the administration of nanomedicines comprising said metal component.

DESCRIPTION OF THE RELATED ART

[0004] The field of nanomedicine utilizes nanoparticles for either the detection or the treatment of diseases. When used to treat diseases, nanoparticles can have several roles: they can be the active pharmaceutical ingredient, the excipient, or the drug carrier [1] Regardless of the application, nanoparticle-based drugs share the common property of being composed of heterogeneous mixtures of nanosized (-1-1000 nm) materials. Many of the nanotechnology -based products have complex formulations such as iron-carbohydrate complexes, liposomes, protein-bound drugs, nanotube -forming drugs and nano emulsions, distinguishing them from traditional small molecule drugs, which have a simple, singular molecular structure [1]. As a result, the terms "complex drug" and, more recently, "non-biological complex drug" (NBCD) have been invoked by some groups to describe nanoparticle based drugs [1-4]

[0005] The heterogeneity of nanoparticle -based drugs has made their development and approval for clinical use a challenge [1,3, 5-7] One key gap lies in identifying which physicochemical properties should be measured to meet regulatory standards [1,3, 5, 7] Nanoparticles are currently regulated in the same manner as small molecule drugs, yet exhibit unique particle size and surface chemistry that is important for their activity. A second gap lies in the breakdown and pharmacokinetics of nanoparticle drugs in vivo. In many instances, indirect in vivo measures have been utilized to evaluate the pharmacokinetics of nanoparticle drugs, because direct measures are not available, leading researchers to infer on the state of the nanoparticles [1].

[0006] One family of nanomedicines which are currently used in the clinic are iron-carbohydrate based nanoparticles [8] . There are six FDA-approved iron-carbohydrate nanoparticle drugs, and for one of these drugs, FERRLECIT (sodium ferric gluconate), a generic version is also FDA approved [1]. Iron- carbohydrate nanoparticle drugs are all administered intravenously (IV), and are utilized to treat iron- deficiency anemia (see, Figures 1A and IB) [9] The goal of the IV iron treatments is to replenish cellular iron so that proteins that are depleted of iron under anemic conditions (and are therefore inactive) are re-activated (effective erythropoiesis) [10] Under normal conditions when cellular iron is depleted, iron is imported into the cell via the transferrin receptor protein (see, Figure 2A) [11,12] This import of iron must be tightly regulated otherwise iron overload occurs. Iron overload results in saturation of the transferrin pathway and any excess iron is then present as“free” or“labile” iron in the plasma. Labile iron can be toxic, as it is imported into cells via non-specific transporters where it can interact with oxygen to form deleterious reactive oxygen species (see, Figure 2B) [13] Thus, it is critical that IV iron replacement therapy provide the appropriate amount of iron at the appropriate rate such that it is delivered into cells via the transferrin import pathway to then be utilized by proteins depleted of iron for erythropoiesis. If iron is released from the drug complex too rapidly, non-specific transporters will be activated leading to toxic consequences. Iron overload-induced toxicity is one of the major serious adverse effects associated with IV iron replacement therapy [14]

[0007] In view of the foregoing, there is a need for the development of new methods that directly measure nanoparticles in vivo·, such methods will enable the development of products containing nanoparticles.

SUMMARY

[0008] The present invention relates to a direct method of determining the amount of metal species present in a sample before and/or after a metal nanoparticle drug has been administered to a subject. The metal species quantified can include the metal nanoparticle drug administered as well as the speciation of other metal species released from the metal nanoparticles in the sample. For example, the method described herein can be used to quantify iron-containing nanoparticles in plasma as well as the speciation of the iron released from said iron-containing nanoparticles in the plasma. The present invention also relates to methods of quantifying metal species present in a sample of the medical product itself (e.g., a vial of sodium ferric gluconate) without the need for any metal-containing nanoparticle to have been administered to a subject.

[0009] In one aspect, a method of separating at least two metal-containing species in a sample and quantifying a concentration of each metal-containing species is disclosed, said method comprising: introducing a sample to a liquid chromatograph (LC) to separate the at least two metal-containing species in the sample into separate fractions; and

detecting each fraction of metal-containing species from the LC using an Inductively Coupled Plasma- Mass Spectrometer (ICP-MS) to quantify the amount of each metal-containing species in the sample.

[0010] In another aspect of the present invention, a method of separating at least two metal-containing species in a sample and quantifying a concentration of each metal-containing species is disclosed, said method comprising:

administering an amount of metal-containing nanoparticles to a subject;

withdrawing the sample from said subject at time x;

introducing a sample to a liquid chromatograph (LC) to separate the at least two metal-containing species in the sample into separate fractions; and

detecting each fraction of metal-containing species from the LC using an Inductively Coupled Plasma- Mass Spectrometer (ICP-MS) to quantify the amount of each metal-containing species in the sample.

[0011] Other aspects, features and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A illustrates the schematic of an iron carbohydrate product.

[0013] Figure IB illustrates the proposed structure of sodium ferric gluconate.

[0014] Figure 2A illustrates iron homeostasis under normal conditions, wherein iron in the plasma is mostly bound to transferrin. Transferrin-bound iron is then imported into the cell via the transferrin receptor protein (TrFl).

[0015] Figure 2B illustrates iron homeostasis under iron overload conditions, wherein transferrin becomes saturated with iron, resulting in downregulation of TrFl. Iron associated with citrate and albumin (i.e., "labile iron") is transported into the cell via non-specific transporters. Once in the cell, the iron can saturate the iron binding sites of intracellular proteins and can react with oxygen, forming toxic reactive oxygen species.

[0016] Figure 3 illustrates the liquid chromatography -inductively coupled plasma-mass spectrometry (LC-ICP-MS) instrumental setup.

[0017] Figure 4 is an ICP-MS chromatogram for counts of iron (56).

[0018] Figure 5 illustrates the validation of the measurement of iron using ICP-MS.

[0019] Figure 6 illustrates the results of the ICP-MS validation method wherein ICP-MS was more accurate at lower concentrations than the ferrozine assays.

[0020] Figure 7A illustrates the LC-ICP-MS analysis of diluted BSA-AuNCs sample.

[0021] Figure 7B illustrates the different molecular weight distribution between the reference BSA, BSA-AuNCs, and size exclusion chromatography fractions of BSA-AuNCs by SDS-PAGE. [0022] Figure 8 illustrates the LC-ICP-MS chromatogram of Fe 56 species (dashed line) and the UV- Vis (280 nm) chromatogram to identify proteins/chromophores (solid line).

[0023] Figure 9 illustrates the LC-ICP-MS chromatogram of Cu 63 species (dashed line) and the UV- Vis (280 nm) chromatogram to identify proteins (solid line).

[0024] Figure 10 illustrates the LC-ICP-MS chromatogram of Zn 66 species (dashed line) and the UV- Vis (280 nm) chromatogram to identify proteins (solid line).

DETAILED DESCRIPTION. AND PREFERRED EMBODIMENTS THEREOF

[0025] The present invention generally relates to a method of quantifying the metal species present in a sample before and/or after a metal nanoparticle drug has been administered to a subject. The metal species quantified can include the metal nanoparticle drug administered as well as the speciation of other metal species produced from the metal nanoparticles in the sample. In one embodiment, the method described herein can be used to quantify iron-containing nanoparticles in a sample as well as the speciation of the iron-containing species in the sample. This method has potential applications not only for iron-containing nanoparticle drugs but also for any nanoparticle or nanomedicine with an inorganic component, i.e., comprises a metal. Advantageously, the method described herein can be used to monitor and measure multiple different metal-containing nanoparticles and/or metal-containing species simultaneously.

[0026] As defined herein, a "particle" corresponds to a species between about 1 nanometer and 10 microns in diameter, including particles or clusters that would be traditionally characterized as nanoparticles or nanoclusters (e.g., about 1 nm to about 100 nm), respectively, and particles or clusters that would be traditionally characterized as micron-sized (e.g., up to about 10 microns). Particle size ranges relevant to the present invention include about 10 nm to about 100 nm, about 10 nm to about 500 nm, about 10 nm to about 1000 nm, about 100 nm to about 500 nm, about 500 nm to about 1000 nm, about 1000 nm to about 5000 nm (5 microns), and about 1000 nm to about 10000 nm (10 microns). A person with an ordinary skill in the art will readily understand that the foregoing particle size ranges are not intended to limit the method described herein and can include larger sizes. Also, it should be appreciated that the particles can be any shape including spherical or a polygonal shape, and can be substantially symmetrical or asymmetrical. Further the material composition of the particles can be organic, inorganic, organometallic, proteinaceous, metallic, polymeric, or any combination of each of these.

[0027] As defined herein, the "particle-containing product" includes a product with nano- and/or micron-sized particles comprising a metal and suspended in a solvent or a mixture of solvents. The particle-containing product can further comprise at least one surfactant, at least one water-soluble organic solvent, at least one dispersant, at least one biocide, at least one buffering agent, at least one pH adjusting agent (e.g., acids and/or bases), as readily determined by the person skilled in the art. The "metal" can be any species including, but not limited to, iron, nickel, zinc, copper, gold, aluminum, silver, platinum, palladium, ruthenium, rhenium, and any combination thereof.

[0028] As defined herein, a "subject" can include any organism including, but not limited to, prokaryotic cells, eukaryotic cells, simple organisms (e.g., C. elegans ), as well as any avian, aquatic, or mammalian species. In one embodiment, the subject is a human.

[0029] As defined herein, liquid chromatography or LC is intended herein to signify high performance liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC) and can include any of the known types of HPLC or FPLC including, but not limited to, normal phase chromatography, reverse phase chromatography, displacement chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and partition chromatography. The type of HPLC used is readily determinable by the person skilled in the art based on what species need to be separated, identified and quantified.

[0030] The current methods for quantitating iron release by intravenous (IV) iron-nanoparticles involves measuring the total iron (TI) and transferrin bound iron (TBI) present in plasma after infusion [15] The amount of non-transferrin bound iron (NTBI) is then inferred by difference : TI = TBI + NTBI. NTBI has traditionally been a catch-all variable intended to represent all iron in plasma that is not TBI. NTBI can include iron bound to the ferritin and albumin proteins (“protein bound iron” or PBI), iron bound to small molecules such as citrate (“labile iron” or LI) and iron still associated with the nanoparticulate drug (“drug bound iron” or DBI). Some of these components, including iron-citrate and iron-albumin, are potentially available for formation of reactive oxidative species (ROS) because iron is loosely bound [16,17] There are some reported methods to directly measure NTBI. These methods typically use chelation (e.g., nitrilotriacetic acid (NT A), desferrioxamine (DFO) and CP851 (a hexadendate pyridine chelator)) or chelation/redox changes (e.g., bleomycin). These methods cannot distinguish between the various iron species present in the plasma, often over-estimate the amount of NTBI and have large errors, limiting their utility and accuracy [18] In addition, none of the reported methods are known to quantify DBf in plasma [19-21]

[0031] Regulatory agencies have a strong interest in approving additional IV nanoparticle drugs, including generics [1,2,5]. However, these approvals are hindered by the absence of robust and accurate methods to measure the fate of the iron-nanoparticle once it is administered [3] A method that allows for the direct measurement of DBI, TBI, and the major species of NTBI (i.e., Fe-citrate, Fe-albumin, Fe-ferritin) would allow for the obtainment of the complete and accurate profile of iron release in a sample, e.g., plasma, after administration of IV iron-nanoparticles. Preferably, this method can be used to quantify metal species present in a sample of the medical product itself prior to administration to a subject.

[0032] In the present invention, a direct method to measure metal-containing nanoparticles along with the other metal-containing species in a sample is disclosed, said method using Liquid Chromatography - Inductively Coupled Plasma-Mass Spectrometry (LC-ICP-MS). In one preferred embodiment, the method relates to a quantitative measure of iron-containing nanoparticles and other iron-containing species in a sample using LC-ICP-MS. In another embodiment, the method relates to a quantitative measure of protein-stabilized gold nanoclusters in a sample using LC-ICP-MS. In still another embodiment, the method relates to a quantification of metal ions in a sample using LC-ICP-MS, wherein said metal ions are selected from the group consisting of iron, nickel, zinc, copper, gold, aluminum, silver, platinum, palladium, ruthenium, rhenium, and any combination thereof.

[0033] In LC-ICP-MS, the ICP-MS is connected to the LC outlet column, with a multiple wavelength detector cell which records the absorbance at a pre-selected UV-visible wavelength and a nebulizer therebetween, wherein the nebulizer produces an aerosol that is compatible with the plasma of the ICP- MS, wherein the LC separates the metal-containing species in the sample and the ICP-MS acts as the detector of said separated metal-containing species. Compared to other detectors, ICP-MS is superior because of its sensitivity and detection limits. Alternative detection systems include, but are not limited to, fluorescence detection and refractive index detection. The process involves first identifying a mobile phase for the LC that is tolerated by the plasma of the ICP-MS as well as an appropriate stationary phase(s), wherein the mobile and stationary phases adequately separate the metal-containing species from one another so that they can be "detected" by the ICP-MS. Calibration curves can be generated using known quantities of said metal-containing species, as readily understood by the person skilled in the art. Using the calibration curves, the concentration of the separated metal-containing species can be determined, as readily understood by the person skilled in the art.

[0034] Using this method, the iron species in a sample, e.g., blood plasma or serum, can be directly measured at some time t following infusion with an iron-nanoparticle drug. These species include TBI (transferrin bound iron), PBI (protein bound iron, e.g., ferritin and albumin), LI (labile iron, e.g., iron citrate) and DBI (drug bound iron, i.e., iron-carbohydrate nanoparticle), wherein total iron (TI) = TBI + PBI + DBI + LI. To the best of the inventors’ knowledge, this is the first report of a direct assay that simultaneously measures the intact IV iron drug (DBI), along with iron bound to target proteins and labile iron. Advantageously, this method has the potential to be applied to iron-nanoparticle drugs as well as other metal-containing nanomedicines in biologically relevant matrices, both before and/or after administration, to aid in the development and approval of novel therapies.

[0035] Accordingly, in a first aspect, a method of separating at least two metal-containing species in a sample and quantifying a concentration of each metal-containing species is disclosed, said method comprising:

introducing a sample to a liquid chromatograph (LC) to separate the at least two metal-containing species in the sample into separate fractions; and

detecting each fraction of metal-containing species from the LC using an Inductively Coupled Plasma- Mass Spectrometer (ICP-MS) to quantify the amount of each metal-containing species in the sample.

[0036] The method of separating at least two metal-containing species can further comprise:

administering an amount of metal-containing nanoparticles to a subject; and withdrawing the sample from said subject at time x,

prior to introducing the sample to the LC.

[0037] In one embodiment of the first aspect, a method of separating at least two iron-containing species in a sample and quantifying a concentration of each iron-containing species is disclosed, said method comprising:

introducing a sample to a liquid chromatograph (LC) to separate the at least two iron-containing species in the sample into separate fractions; and

detecting each fraction of iron-containing species from the LC using an Inductively Coupled Plasma- Mass Spectrometer (ICP-MS) to quantify the amount of each iron-containing species in the sample.

[0038] In another embodiment of the first aspect, a method of separating at least two iron-containing species in a sample and quantifying a concentration of each iron-containing species is disclosed, said method comprising:

introducing the sample to a liquid chromatograph (LC) to separate the at least two iron-containing species in the sample into separate fractions; and

detecting each fraction of iron-containing species from the LC using an Inductively Coupled Plasma- Mass Spectrometer (ICP-MS) to quantify the amount of each iron-containing species in the sample, wherein the iron-containing species are selected from the group consisting of transferrin bound iron (TBI), protein bound iron (PBI), labile iron (LI), drug bound iron (DBI), and combinations thereof.

[0039] In still another embodiment, a method of separating at least two iron-containing species in a sample and quantifying a concentration of said iron-containing species is disclosed, said method comprising:

administering an amount of iron-containing nanoparticles to a subject; and

withdrawing the sample from said subject at time x,

introducing the sample to a liquid chromatograph (LC) to separate the at least two iron-containing species in the sample into separate fractions; and

detecting each fraction of iron-containing species from the LC using an Inductively Coupled Plasma- Mass Spectrometer (ICP-MS) to quantify the amount of each iron-containing species in the sample, wherein the iron-containing species are selected from the group consisting of transferrin bound iron (TBI), protein bound iron (PBI), labile iron (LI), drug bound iron (DBI), and combinations thereof.

[0040] Advantageously, the method of the first embodiment can be performed to quantitate the concentration of in vivo metal-containing species, and preferably in vivo iron-containing species, not previously determinable using a direct method. The method can be performed without having to introduce any oxidizing or reducing species to the sample prior to analysis or having to use any oxidation-sensitive probes. The "sample" can include any solution that includes a metal, preferably a “biological solution” including, but not limited to, blood, blood plasma, blood serum, cells, stool, sputum, bronchoalveolar lavage fluid (BALF), urine, saliva, amniotic fluid, breast milk, or any combination thereof. Alternatively, the sample can be the metal-containing nanoparticle or nanocluster solution, e.g., a metal-containing nanoparticle or nanocluster drug devoid of the aforementioned biological solutions, prior to administration. Exemplary modes of administration include intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, oral, or intraocular injections. In one embodiment, the mode of administration is intravenous. In another embodiment, the mode of administration is oral. The quantification of each metal-containing species is done using calibration curves, as readily understood by the person skilled in the art. The time x can be any time from 1 min to 10 weeks, as necessary to understand the speciation of metal, e.g., iron, in the sample. It should be appreciated that a sample can be withdrawn from the subject prior to administration of the metal-containing nanoparticles (e.g., at time zero) and the metal speciation determined for comparison to the metal speciation at time x. In one embodiment, the metal quantified herein is iron. In another embodiment, the metal quantified is gold. In still another embodiment, the metal quantified is zinc. In yet another embodiment, the metal quantified is copper. In another embodiment, the metal quantified is manganese.

[0041] The species present in samples that can bind a metal can differ significantly in molecular weight relative to the administrated metal-containing nanoparticle, therefore liquid chromatographic technique size exclusion chromatography (SEC) can be utilized. For example, in plasma, the species transferrin, albumin, ferritin, and citrate can bind iron, each having a significantly different molecular weight than the iron-containing nanoparticle sodium ferric gluconate (between 0.43 and 450 kDa) (see, Table 1). SEC separates molecules by size where the pore size of the stationary phase limits the interactions between analytes and the stationary phase of the column. Importantly, SEC preserves proteins in their native folded, and metal coordinated states [22,23] Accordingly, in a preferred embodiment, the LC used is an SEC -type LC.

Table 1: Iron speciation in plasma for a person treated with sodium ferric gluconate.

a: Ref. [25]; b: Ref : [26]; c: Ref. [27]; d: Ref. [17]; e: Ref. [7]; f: Ref. [24] [0042] Preferably, in the assay method described herein, samples are first injected onto an HPLC equipped with at least one SEC column, or in the alternative at least two SEC columns in series. The eluted solution then passes through a multiple wavelength detector cell which records the absorbance at a pre-selected UV-visible wavelength, as readily determined by the person skilled in the art. The eluted solution can then be flowed into a nebulizer and directly into the plasma of the ICP-MS for metal analysis and subsequent quantification. The time of resolution on the LC, as measured using UV-visible spectroscopy, substantially mimics the time of ionization on the ICP-MS, because of the immediate introduction from the LC to the ICP-MS, thereby allowing the user to identify which metal species, e.g., iron, is being quantified.

[0043] In one embodiment, the species being quantified comprises iron and the pre-selected UV- visible wavelength is about 280 nm. In another embodiment, the species being quantified comprises iron and the pre-selected UV-visible wavelength is about 220 nm.

[0044] With regards to the SEC columns, preferably the columns can separate molecules with a wide molecular weight range (5,000-1,250,000 Da) in a short run time (< 10 min). Example columns include, but are not limited to, the Agilent SEC-5 (5 pm, 300 A, 4.6 x 150 mm column), the Agilent Bio SEC -3 (3 pm, 300 A, 4.6 mm x 300 mm column), the Agilent Bio SEC-3 guard column (3 pm, 300 A, 4.6 x 50 mm), the AdvancedBio SEC column (2.7 pm, 300 A, 4.6 x 50 mm), or the equivalent thereof. Other useful columns include, but are not limited to, Sephacryl or XK or Mono Q (GE Healthcare Life Sciences), SynChronpak, and FRACTOGEL (EMD Millipore). For example, it was determined that all of the iron-containing species of interest could be chromatographically resolved when two Agilent SEC- 3 columns were placed in series, preceded by an Agilent Bio SEC-3 guard column. It should be appreciated that the column or columns can be readily determined by the person skilled in the art based on the knowledge of the species to be resolved. For example, one column may be used. Alternatively, two or more columns that may be the same as or different from one another may be used. When two or more columns are selected they are preferably placed in series.

[0045] Regarding the mobile phases, in a preferred embodiment a neutral buffer is used, e.g., 10 mM Tris having a pH of 7.4. Other contemplated mobile phases include, but are not limited to, HEPES, phosphate buffers, phosphate buffered saline, ammonium acetate buffer, CHAPS, MOPS, and MES, all at approximately neutral pHs. Flow rates are readily determined by the person skilled in the art. In one embodiment, the flow rate is 0.4 mL/min. The volume of the sample injected can be in a range from about 10 pL to about 100 pL.

[0046] The first direct method to quantify metal-containing nanomedicines, e.g., iron-nanoparticle drugs, in biological media is disclosed herein. The method, which couples LC with ICP-MS, allows for the simultaneous quantification of iron distribution in a sample, e.g., plasma or serum, after administration of iron-nanoparticle drugs. Advantageously, the disclosed method is the first known method to directly measure all of the iron species (i.e., LI, DBI, PBI, TBI) at the same time. The method provides a reliable quantification of said iron-containing species without having to introduce any oxidizing or reducing species to the sample prior to analysis or having to use any oxidation-sensitive probes. Further, the ability to measure different iron species permits the measurement of iron biomarkers as indicators of specific diseases, e.g., Heme oxygenase (HO-1) is a biomarker of increased oxidative stress. The method can also be used to test for product stability over time. This strategy has applications for the evaluation of new iron-nanoparticle drugs, both innovator and generic, as well as more broadly for metal-based nanoparticles (e.g., gold nanoparticles) used in biomedical sciences and in non-biological complex drug development.

[0047] Accordingly, in another aspect, a method of comparing the physico-chemical differences between a first iron-containing nanoparticle product and a second iron-containing nanoparticle product, said method comprising: (i) analyzing the first iron-containing nanoparticle product using LC-ICP-MS to measure one or more iron species contained in the first iron-containing nanoparticle product; (ii) analyzing the second iron-containing nanoparticle product using LC-ICP-MS to measure one or more iron species contained in the second iron-containing nanoparticle product; and (iii) comparing the first iron-containing nanoparticle product to the second iron-containing nanoparticle product, wherein a difference between the first iron-containing nanoparticle product and the second iron-containing nanoparticle product corresponds to a difference in the physico-chemical makeup between the first and the second iron-containing nanoparticle products. It should be understood that the first iron-containing nanoparticle product can be a batch of product at time zero and the second iron-containing nanoparticle product can be from the same batch at a time /, to verily product stability. The person of skill in the art would understand that the measurements at time zero would be provided to the user measuring at time t. Alternatively, the first iron-containing nanoparticle product can be an innovator product and the second iron-containing nanoparticle product can be a generic of the innovator.

[0048] In still another aspect, a method of using LC-ICP-MS to analyze and quantify protein-stabilized gold nanoclusters is described herein. Protein-stabilized gold nanoclusters are a new type of nano-sized molecule that exhibit colorimetric changes upon protein activity. There is interest in developing these nanoclusters as novel detection agents for diseases, however, methods to analyze their purity and activity have been lacking to date. Using the LC-ICP-MS apparatus and the method described herein, protein-stabilized gold nanoclusters, specifically insulin stabilized gold nanoclusters, can be purified and the gold:protein stoichiometry determined. For example, the gold content can be quantified using ICP-MS while the protein can be quantified using UV-vis. The method will have relevance for the development of gold nanoclusters to detect diseases (e.g., antibody gold nanoclusters) or for use as imaging agents.

[0049] The features and advantages of the invention are more fully shown by the illustrative examples discussed below.

Exam nlc 1 [0050] A protein solution containing thyroglobulin, y-globulin, albumin, ribonuclease A and para- aminobenzoic acid standards (molecular weights: 670 to 0.14 kDa) was used to calibrate a column setup for size determination, wherein the column setup comprises two Agilent SEC -3 columns placed in series, preceded by an Agilent Bio SEC -3 guard column. Solutions of transferrin, sodium ferric gluconate, albumin and iron citrate were spiked into plasma to identify where each species eluted in a plasma matrix (see, Table 1) [7,17,24-27] Sodium ferric gluconate eluted first (9 - 10 min) followed by iron-ferritin (10 - 10.5 min), iron-albumin (10.5 - 11.5 min), iron-transferrin (11.5 - 13.5 min) and iron-citrate (15 - 16.5 min) (see, Table 1). To independently confirm our peak identification, the peaks were collected and their molecular weights were confirmed by MALDI-MS or ESI-MS (not shown). The validity of this analytical method was verified per FDA guidelines for bioanalytical method validation [28]

[0051] With regards to data processing for PBI, TBI, and LI concentrations, a calibration curve was constructed from known concentrations of holo-transferrin. The peak areas of the calibration samples versus the peak area of the internal standard were analyzed using linear regression. The sample PBI, TBI, and labile iron concentrations were determined by comparing the peak area of each species versus the peak area of the internal standard to the prepared calibration curve.

[0052] With regards to data processing for DBI concentrations, a calibration curve was constructed from known concentrations of sodium ferric gluconate (SFG) spiked into plasma. The peak areas of the calibration samples versus the peak area of the internal standard were analyzed using linear regression. The sample DBI concentrations were determined by comparing the peak area of each species versus the peak area of the internal standard to the prepared calibration curve.

Exam iilc 2

[0053] The LC-ICP-MS assay was applied to a clinically relevant sample. A healthy volunteer was administered 125 mg of sodium ferric gluconate (SFG) and the volunteer’s blood plasma sampled at times 0 (zero) and 3 h after dose was evaluated. The column setup comprises two Agilent SEC -3 columns placed in series, preceded by an Agilent Bio SEC -3 guard column. The mobile phase was 10 mM Tris pH 7.4 with a flow rate of 0.4 mL/min and the injection volume was 10 pL. Figure 4 shows the resulting LC-ICP-MS chromatograms. The chromatogram in Figure 4 shows the volunteer’s native iron distribution prior to sodium ferric gluconate (SFG) IV iron treatment as well as the volunteer’s plasma 3 h after infusion. The first peak in the spectrum (~ 1 min) is the internal standard which is added post column. The next peak, which appears around 11 min is protein bound iron (PBI, Fe-albumin and Fe-ferritin). This is followed by the largest peak, TBI, around 12 min, followed the less abundant peak LI (Fe-citrate) at 16 min. Two key features are noted in the post-infusion plasma: a new peak at 10 min for DBI and increased in the TBI and LI peaks as compared to the pre-infusion levels. Quantification of DBI yielded plasma levels of 3,500 parts per billion (ppb) at 3 h after a 125 mg SFG dose. The increase in TBI and LI observed was similarly quantified with levels of iron-bound transferrin of 787 ppb at 0 h and 1680 ppb at 3 h and levels of labile iron of 69 ppb at 0 h and 102 ppb at 3 h after a 125 mg SFG dose. Together, these data provide compelling evidence that the LC-ICP-MS can be applied to clinically relevant samples, and are the first example of directly measuring DBI in plasma.

Example 3

[0054] The method of measuring the total iron concentration using ICP-MS was validated as follows. Plasma stock was spiked with sodium ferric gluconate (SFG) in concentrations of 0.2, 2, 5, 10, 20, and 40 ppm to form a spiked plasma solution as indicated in Figure 5. 200 pL of each spiked plasma solution was combined with 800 pL of water, and 500 pL of HNO3 (cone) and heated for 12 h at 80°C. Thereafter 4 mL of ¾0 was added and the concentration of iron analyzed using ICP-MS. The results are shown in Figure 6, wherein the original amount added, the concentration determined using ICP-MS and the concentration determined using a ferrozine assay are shown. It can be seen that ICP-MS measures showed greater accuracy at lower concentrations compared to the ferrozine assay.

Example 4

[0055] The one-pot synthesis of gold containing protein-stabilized nanoclusters (AuNCs) gives a final product which is a mixture of multiple species that contain varying amounts of gold. AuNC’s have been utilized as innovative imaging agents and sensors due to their unique properties. In this experiment, LC- ICP-MS was used to purify the AuNC mixture to 1) understand the different species that were produced during the reaction and 2) test the individual species properties to understand if one of them is a more efficient sensor or imaging agent.

[0056] To demonstrate the efficacy of the method described herein, gold-containing BSA-stabilized nanoclusters (AuNCs) were separated high performance liquid chromatography (HPLC) equipped with size exclusion column (SEC) was directly coupled with an inductively coupled plasma-mass spectrometer (Agilent 7700X ICP-MS, Agilent Technologies, Santa Clara, California). Stocks of AuNCs were diluted by 10 mM ammonium acetate at pH 7.4 and injected in an Agilent 1260 HPLC equipped with a BIO SEC -3 Guard column (3 pm 300 A, 4.6 x 50 mm) and an AdvancedBio SEC column (2.7 pm, 300 A, 4.6 x 50 mm) in series. Eluent from HPLC was directly injected into the ICP- MS via a micromist nebulizer and the concentration of gold ( ¹⁹⁷ Au) in AuNCs samples was measured. The ICP-MS parameters used for analysis were: an RF power of 1550 W, and helium gas flow of 4.3 mL/min, an argon gas flow of 0.99 L/min, and octopole RF of 190V, and an OctP bias of - 18V. Data analysis was performed via Agilent’s Mass Hunter Software.

[0057] The LC-ICP-MS analysis of diluted BSA-AuNCs sample is shown in Figure 7A. It can be seen that a majority of ¹⁹⁷ Au was detected at 12-15 minutes of retention time.

[0058] Figure 7B illustrates the molecular weight distribution between the reference BSA, BSA- AuNCs, and size exclusion chromatography fractions of BSA-AuNCs by SDS-PAGE. Lanes 1-3 show the protein ladder, BSA and the Au-BSA complex (pre-column), the subsequent lanes correspond to elution time (11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes). Figure 7B evidences that the BSA-AuNCs can be separated using the method described herein.

Example 5

[0059] The LC-ICP-MS assay developed to measure iron speciation (and iron nanoparticle drug) was applied to mouse plasma and serum samples. The samples were from mice that had been administered different concentrations of iron (normal iron diet, low iron diet and high iron diet), and were of different ages and sex.

[0060] The LC-ICP-MS assay was also applied to measure multiple metals at once. In addition to iron, zinc, copper and manganese were separated and quantified. Similarly, these studies were conducted in both serum and plasma.

[0061] Mouse serum samples (80 pL) were diluted with 10 mM Tris (320 pL), transferred to Coming Costar Spin-X centrifuge tube filters (cellulose acetate membrane, pore size 0.22 pm), and centrifuged at 14,000 x g for 5 minutes. The samples were then transferred to HPLC vials equipped with 200 pL inserts and analyzed by LC-ICP-MS. Iron LC-ICP-MS quantification was performed on an Agilent 7700x ICP-MS (Agilent Technologies, Santa Clara, CA, USA). Iron ( ⁵⁶ Fe) concentrations were detected using an Octopole Reaction System cell (ORS) in He mode to remove any interferences. The ICP-MS parameters used for the analysis were: an RF power of 1550 W, an argon carrier gas flow of 0.99 L/min, helium gas flow of 4.3 mL/min, octopole RF of 190 V, and OctP bias of -18 V. Samples eluted from the HPLC columns were directly infused into the ICP-MS using a micromist nebulizer. The peri pump on the ICP-MS was utilized to continually flow the internal standard solution (100 ppb transferrin) from our stock solution container to the LC. This solution flowed to the tCP-MS, post column, via a LC valve switch at the beginning of the run. The internal standard solution monitored any ICP-MS instrumental shift during the runs. After 30 sec, the valve was then switched back, so the column elution flowed to the ICP-MS for metal quantification. The integration was performed for peak area within the following elution windows: Internal standard: 1-1.5 min, ferritin and albumin (PBI) 8.8-11 min, TBI 11.8-14.2 min and LI 15-18 min. While performing the iron quantification we also monitored zinc ( ⁶⁶ Zn), manganese ( ⁵⁵ Mn) and copper ( ⁶³ Cu) signals, so we were able to visualize the individual metal speciation. Unfortunately, without a calibration curve of know concentration for the individual metals we cannot quantify the metal concentrations for each species. That said, it is well within the knowledge of the person skilled in the art to prepare a calibration curve for each metal.

[0062] Referring to Figure 8, it can be seen that the method of the present disclosure successfully separated the different iron species contained in the mouse serum samples and it will be understood by the person skilled in the art that concentrations can be determined if a calibration curve is prepared. Figures 9 and 10 show the successful separation of the copper and zinc species, respectively. [0063] Although the invention has been variously disclosed herein with reference to illustrative embodiments and features, it will be appreciated that the embodiments and features described hereinabove are not intended to limit the invention, and that other variations, modifications and other embodiments will suggest themselves to those of ordinary skill in the art, based on the disclosure herein. The invention therefore is to be broadly construed, as encompassing all such variations, modifications and alternative embodiments within the spirit and scope of the claims hereafter set forth.

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