Snapshots of Iron Speciation: Tracking the Fate of Iron Nanoparticle

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Snapshots of Iron Speciation: Tracking the Fate of Iron Nanoparticle Drugs via a Liquid ChromatographyInductively Coupled Plasma-Mass Spectrometric Approach. Heather M. Neu, Sergei A. Alexishin, Joel E. P. Brandis, Anne M. C. Williams, Wenjing Li, Dajun Sun, Nan Zheng, Wenlei Jiang, Ann Zimrin, Jeffrey C. Fink, James E Polli, Maureen A Kane, and Sarah L. J. Michel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01215 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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Molecular Pharmaceutics

Snapshots of Iron Speciation: Tracking the Fate of Iron Nanoparticle Drugs via a Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Approach. Heather M. Neu , Sergei A. Alexishin , Joel E. P. Brandis , Anne M. C. Williams , Wenjing Li , 1

1

1

1

1

Dajun Sun , Nan Zheng , Wenlei Jiang , Ann Zimrin , Jeffrey C. Fink , James E. Polli , Maureen A. 2

2

2

3

4

1

Kane , Sarah L. J. Michel 1

1*

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore,

1

MD 21201, USA Office of Research and Standards, Office of Generic Drugs, Center for Drug 2

Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, USA Oncology program, University of Maryland School of Medicine, Baltimore, MD 21201, 3

USA Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 4

21201, USA

*

email: [email protected]

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Nanomedicines are nanoparticle-based therapeutic or diagnostic agents designed for targeted delivery or enhanced stability. Nanotechnology has been successfully employed to develop various drug formulations with improved pharmacokinetic characteristics, and current research efforts are focused on the development of new innovator and generic nanomedicines. Nanomedicines, which are often denoted as complex or non-biological complex drugs, have inherently different physicochemical and pharmacokinetic properties than conventional small molecule drugs. The tools necessary to fully evaluate nanomedicines in clinical settings are limited, which can hamper their development. One of the most successful families of nanomedicines are iron-carbohydrate nanoparticles, which are administered intravenously (IV) to treat iron-deficiency anemia. In the U.S, the FDA has approved six distinct iron carbohydrate nanoparticles, but only one generic version (sodium ferric gluconate for Ferrlecit). There is significant interest in approving additional generic iron-carbohydrate drugs; however, the lack of a direct method to monitor the fate of the iron nanoparticles in clinical samples has impeded this approval. Here we report a novel Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry (LC-ICP-MS) method that allows for the direct quantification of the iron-carbohydrate drugs in clinical samples, while simultaneously measuring the speciation of the iron released from the nanoparticles in biological samples. To our knowledge, this is the first time that iron nanoparticles have been observed in clinical samples, opening the door for direct pharmacokinetic studies of this family of drugs. This method has potential applications not only for iron-nanoparticle drugs but also for any nanomedicine with an inorganic component.


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Keywords Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry (LC-ICP-MS) Sodium Ferric Gluconate (SFG) Drug Bound Iron (DBI) Nanomedicine Labile Iron (LI) Non-transferrin bound iron (NTBI) Protein bound iron (PBI) Metal Speciation Complex Non-Biological Drug



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1. Introduction The field of nanomedicine utilizes nanoparticles for either the detection or 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. Regardless of their application, 1

nanoparticle-based drugs share the common property of being nanosized (~1-1000 nm) materials, and many nanomedicines containing complex active ingredients (e.g., iron-carbohydrate complexes, liposomes, protein-bound drugs) which distinguishes them from traditional small molecular drugs, which have a simple, singular molecular structure. Although ‘nanoparticle drugs’ 1

are not categorized separately by the FDA, based upon these properties, 51 FDA approved drugs can be considered nanoparticle-based. Recently, the terms ‘complex drug’ and ‘non-biological 2

complex drug’ (NBCD) have been invoked by some groups to describe nanoparticle-based drugs.

1,

3-6

The heterogeneity in structure and/or function of nanoparticle-based drugs has made their development and approval for clinical use a challenge.

1, 3, 5, 7-8

The physicochemical properties

(molecular weight distribution, particle size, iron oxidation state, iron concentrations, ect.) of these drug products have been extensively characeterized. However, a key gap lies in identifying which 1, 9

physicochemical properties inform on in vivo acitivy and therefore should be measured to meet regulatory standards.

1, 5, 7

The FDA has recently developed a guidance for drug products containing

nanomaterials. Such guidance reflects the unique particle size and surface chemistry properties of 9

nanoparticles that can impact activity. However, nanoparticles are currently regulated in the same manner as small molecule drugs. 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,


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leading researchers to infer the state of the nanoparticles in vivo. Development of new methods 1, 10

that directly measure nanoparticles in vivo is optimal, and will enable the development of drug products containing nanomaterials.

3

One family of nanomedicines that are currently used clinically are iron-carbohydrate based nanoparticles. There are six FDA-approved iron-carbohydrate nanoparticle drugs, and for one of 11

these drugs, Ferrlecit, a generic version (sodium ferric gluconate) is also FDA approved. Iron1

carbohydrate nanoparticle drugs are all administered intravenously (IV), and are utilized to treat iron-deficiency anemia. (Fig. 1) 1

Figure 1. A cartoon of an iron carbohydrate product (Left) and the proposed structures of sodium ferric gluconate (Right).

The goal of the IV iron treatments is to replenish cellular iron so that proteins that are deplete in iron under anemic conditions (and are therefore inactive) are re-activated (effective erythropoiesis). Under normal conditions when cellular iron is depleted, iron is imported into the 12

cell via the transferrin receptor (Fig. 2 left).

13-15

Upon administration, nanoparticle-enclosed iron

from parenteral products is processed by the reticuloendothelial system, loaded onto transferrin, and transported to tissues. It is also important to address that a small fraction of iron in intravenous formulations exists as a more labile form, which is typically readily scavenged by circulating transferrin.

16-17

A rapid influx of considerable amounts of iron can saturate circulating transferrin’s 5 ACS Paragon Plus Environment


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iron binding sites, causing iron overload and any excess iron is then present as “free” or “labile” iron in the plasma.

14, 18-21

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 (Fig. 2 22

right). Thus, it is critical that IV iron replacement therapy provide the appropriate amount of iron such that it is imported into cells via the transferrin import pathway to then be utilized by iron depleted proteins.

23-25

If too much IV iron is delivered, non-specific transporters will be activated

leading to toxicity. Iron overload-induced toxicity is one of the major serious adverse effects associated with IV iron replacement therapy.

20

Figure 2. Cartoon diagram of iron homeostasis under normal and iron overload conditions. Left, under normal conditions, iron in the plasma is primarily bound to transferrin (blue cartoon). Transferrin bound iron is imported into cells via the transferrin receptor protein (TrF1). Upon internalization, TrF1 releases ferric iron inside the endosome, where it is reduced by Steap3 metalloreductase. Ferrous iron is then shuttled into the cytoplasm via divalent metal transporter 1 (DMT1) and type IV mucolipidosis-associated protein (TRPML1). From there, iron is either stored by incorporation into ferritin, utilized by Fe-dependent molecules/processes, or exported out of the cell via ferroportin. In the extracellular space, ferrous iron is rapidly oxidized back to its ferric 6 ACS Paragon Plus Environment

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form by hephaestin and recycled by associating with apo transferrin. Right, under iron overload conditions, transferrin becomes saturated with iron, resulting in downregulation of TrF1. The excess iron is bound to citrate and albumin, also called ‘labile iron’ and the iron is transported into the cell via non-specific metal transporters, DMT1 and Zip14. Once in the cell, the excess iron saturates iron binding sites of intracellular proteins. Elevated Fe concentrations also upregulate hepcidin production, which inhibits ferroportin-mediated iron export. Unable to leave the cell, remaining iron (i.e. labile iron) accumulates in the cytoplasm, where it can react with cellular oxygen to form toxic reactive oxygen species. The current methods for quantitating iron release by IV iron nanoparticles involves measuring the total iron (TI) and transferrin bound iron (TBI) present in plasma after infusion. The amount of 26

‘non-transferrin bound iron’ or NTBI is then inferred by difference: TI = TBI + NTBI. NTBI is a catch-all variable intended to represent all iron in plasma that is not TBI. NTBI can include iron bound to the proteins ferritin and albumin (protein bound iron or PBI) and iron bound to small molecules such as citrate (labile iron or LI).

19, 27

Some of these components - Iron-citrate and iron-

albumin are potentially available for formation of reactive oxidative species – because iron is loosely bound. There are some reported methods to directly measure NTBI. These methods 27

typically use chelation [e.g. nitrilotriacetic acid (NTA), desferrioxamine (DFO) and CP851 (a hexadendate pyridine chelator) or chelation/redox changes (e.g. bleomycin)].

28-31

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. Notably, during early 28

stages of infusion, some iron is predicted to remain as the nanoparticulate drug (drug bound iron or DBI). None of these methods utilized measure and quantify this drug bound iron species, DBI. 32-34

28,

Therefore, very little is known about the direct pharmacokinetics of DBI. The development of

a direct measure of the drug in plasma (DBI) would be a major advance.

A direct method that allows for the simultaneous measurement of DBI, TBI and the major species of NTBI (e.g. Fe-citrate, Fe-albumin, Fe-ferritin) would allow for the complete and accurate 7 ACS Paragon Plus Environment


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profile of iron release in plasma after administration of IV iron nanoparticles to be obtained. Regulatory agencies have a strong interest in approving additional IV nanoparticle drugs, including generics.

1, 3, 7

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. Herein, we present a direct 5

method to measure iron nanoparticles (DBI) along with all of the other iron species present in plasma using Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry (LC-ICPMS). These species are DBI (drug bound iron), TBI (transferrin bound iron), PBI (protein bound iron – ferritin and albumin) and LI (labile iron – including iron citrate), such that total iron TI = DBI + TBI + PBI + LI. The method has been rigorously validated, per FDA guidance, for use in clinical samples. We also present data in which we analyzed the plasma from volunteers who had been administered the IV-iron drug, as proof of concept that the method can be applied to clinical samples. To the best of our knowledge, this is the first report of the application of LC-ICP-MS to simultaneously measure and quantify the intact IV iron drug (DBI), along with iron bound to target proteins (TBI and PBI), and labile iron (LI) in plasma. This method has the potential to be applied to both iron nanoparticle drugs and to metal containing nanomedicines in biologically relevant matrices, to aid in the development and approval of novel therapies.

MATERIALS AND METHODS Preparation of Tris Buffer (10 mM, pH 7.4). Tris Base (2-Amino-2-(hydroxymethyl)-1,3propanediol, 4.85 g, Fisher BioReagents) was dissolved in Milli-Q water (4 L) and then titrated to pH 7.4 with 6 M hydrochloric acid (trace metal grade, Fisher Chemical). This solution was used as the mobile phase for the LC-ICP-MS.


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Preparation of holo-transferrin calibration curve and internal standard. Crystallized human holo-transferrin (Sigma-Aldrich) was used to prepare a transferrin bound iron calibration curve to quantify TBI, PBI and LI in human plasma. A stock solution (stock 1) was prepared by dissolving 0.036 g of holo-transferrin in 10 ml of 10 mM Tris buffer and mixing via vortex. The protein stock was kept on ice. A calibration curve consisting of 11 points was prepared from serial dilutions of stock 1 with 10 mM Tris ranging from 100 – 5000 ppb. An aliquot (300 µL) of each calibration sample was transferred to Corning Costar Spin-X centrifuge tube filters (cellulose acetate membrane, pore size 0.22 μm), and then centrifuged at 14,000 x g for 5 minutes. The samples were then transferred to HPLC vials equipped with 200 µL inserts. To prepare the 100 ppb holotransferrin internal standard solution, 1 mL of stock 1 was diluted with 49 mL of 10 mM Tris.

Preparation of sodium ferric gluconate (SFG) calibration curve. For the calibration curve for DBI, Ferrlecit (Sanofi-Aventis) – abbrevieated as SFG - spiked into plasma was utilized. Prepared plasma was collected from the University of Maryland Medical Center blood bank and combined to ensure a uniform plasma solution. The plasma solution was separated into conical tubes and stored at - 20°C. Prior to calibration and sample preparation, the plasma was thawed under warm water and thoroughly mixed via vortex. To prepare the DBI calibration curve, a new vial of Ferrlecit was opened and a calibration curve consisting of 9 points was prepared from serial dilutions of Ferrlecit ranging from 12.5 – 12,500 ppm with 10 mM Tris. The prepared diluted stocks of SFG (50 µL) were then spiked into the thawed plasma (350 µL). Aliquots (80 µL) of the prepared SFG calibration samples were diluted with 10 mM Tris (320 µL), transferred to Corning Costar Spin-X centrifuge tube filters (cellulose acetate membrane, pore size 0.22 μm), and



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centrifuged at 14,000 x g for 5 minutes. The samples were then transferred to HPLC vials equipped with 200 µL inserts.

Plasma samples preparation. All human plasma samples were obtained and used in compliance with protocols approved by the University of Maryland, Baltimore (UMB) Institutional Review Board (IRB). At each scheduled time point, blood was collected in 4 mL heparinized tubes. Each tube was then immediately placed on ice and then centrifuged (2000 rmp, 4 °C, 10 minutes) within 15 minutes of collection to produce plasma. The plasma was then removed, split into 500 µL alliquots, quickly frozen on dry ice, and stored at - 80°C. Prior to sample preparation, frozen plasma was thawed at RT (24 ºC) and thoroughly mixed via vortex. Aliquots (80 µL) of plasma were diluted with 320 µL of 10 mM Tris buffer at pH 7.4, vortexed, transferred to Corning Costar Spin-X centrifuge tube filters (cellulose acetate membrane, pore size 0.22 μm), and centrifuged at 14,000 x g for 5 min. The samples were then transferred to LC vials equipped with 200 µL plastic inserts. Samples were kept at 4 °C to promote analyte stability.

Liquid chromatography (LC). All iron species present in plasma were separated by size exclusion chromatography (SEC) utilizing two Agilent Bio SEC-3 LC columns (3 μm, 300 Å, 4.6 mm x 300 mm) and a Bio SEC-3 guard column (3 μm, 300 Å, 4.6 mm x 50 mm) in series. The samples were injected onto an Agilent 1260 Infinity LC equipped with an Agilent 1260 Infinity Bio-inert Quaternary Pump, an Agilent 1260 Infinity Bio-inert High-Performance Autosampler maintained at 4 °C via an Agilent 1290 Infinity Thermostat, an Agilent 1290 Infinity Thermostatted Column Compartment maintained at 25 °C, and an Agilent 1260 Infinity Multiple 10 ACS Paragon Plus Environment

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Wavelength Detector VL. The mobile phase was 10 mM Tris (pH 7.4) at 0.4 mL/min, the injection volume was 10 µL, and the total run time was 20 min.

Inductively coupled plasma mass spectrometry (ICP-MS). Iron quantification was performed on an Agilent 7700x ICP-MS (Agilent Technologies, Santa Clara, CA, USA). Iron 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 ICPMS 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 ICP-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, DBI 8.7-12 min, ferritin and albumin (PBI) 8.8-11 min, TBI 11.814.2 min and LI 15-18 min.

MALDI-MS Analysis of TBI and PBI. To confirm the identity of the native iron proteins present in the plasma, each protein peak was separated via LC as described, collected, and lyophilized to dryness (Savant SpeedVac concentrator). A matrix solution (30 mg/mL Sinapinic acid, 0.3 % TFA, 60% Acetonitrile and 40% water) was added to each sample, after which the samples were 11 ACS Paragon Plus Environment


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transferred to a steel target plate and allowed to evaporate to dryness. The samples were then analyzed on a Bruker UltrafleXtreme MALDI TOF/TOF Mass Spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a smartbeam II Nd:YAG laser, 355 nm, operated in linear positive ion mode. The laser fluence was optimized for each sample using a frequency of 500 Hz and summing 500-1000 shots. Data was analyzed in FlexAnalysis version 3.4 (Bruker Daltonics, Bremen, Germany).

ESI-MS Analysis of LI. To identify the low molecular weight species, present in plasma, plasma was separated by LC as described and the species that eluted at 15 - 16.5 minutes, was collected from 15 – 16.5 min and then infused at 10 µL/min into a Waters Synapt G2S quadrupole-time of flight (Q-TOF) mass spectrometer using electrospray ionization (ESI) in negative ion mode. Source conditions were as follows: source temperature 50 C, capillary voltage 1.5 kV. Acquisition o

time was 1 min and data are displayed as averaged spectra. Iron citrate (500 ppm) in 10 mM ammonium acetate buffer was used as a positive control. Data were acquired and analyzed using MassLynx version 4.1 (Waters Corporation, Milford, MA).

RESULTS AND DISCUSSION Development of LC-ICP-MS Assay to simultaneously and directly measure DBI, TBI, PBI and LI in Plasma. In order to quantify all of the species present in plasma after infusion with an iron nanoparticle drug, we needed to develop a robust separation/detection technique. In recent years, an approach in which liquid chromatography (LC) is coupled to inductively coupled plasma mass spectrometry (ICP-MS) has been shown to be applicable to the separation of native iron 12 ACS Paragon Plus Environment

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proteins and iron complexes in cells and plasma.

35-40

Encouraged by these reports, we sought to (1)

develop LC-ICP-MS such that separation of the iron nanoparticle drug (DBI) from native iron proteins and iron complexes in plasma could be achieved, (2) rigorously validate the method, per FDA guidance, such that it could be applied to clinical samples and (3) apply the assay to a clinical 41

sample to demonstrate ‘proof of concept.’

Separation of Iron Species in Plasma via Liquid Chromatography. To develop the LC method, we chose to utilize size exclusion chromatography (SEC) as our chromatographic material, because the species present in plasma that can bind iron (e.g. transferrin, albumin, ferritin, citrate) along with the iron drug, sodium ferric gluconate (DBI, SFG), differ significantly in molecular weight (between 0.43 and 450 kDa) (Table 1).

1, 9

As SEC separates molecules by size and,

importantly, preserves proteins in their native folded and metal coordinated states, we reasoned that we could separate all species by size in their active forms.

36,42-43

The LC was coupled to an

Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) as well as an in-line UV-visible detector (Fig. 3) to quantify iron and detect the species to which it was coordinated. We initially ran the LC-SEC with just plasma (no added drug) to determine if the SEC-ICP setup was suitable for our application. The plasma samples were injected onto an Agilent 1260 Infinity LC equipped with two SEC columns in series. (Agilent Bio SEC-3 (3 μm, 300 Å, 4.6 mm x 300 mm)) preceded by an Agilent Bio SEC-3 (3 µm, 300Å, 4.6 x 50 mm) guard column. The eluted solution was passed through a multiple wavelength detector cell which recorded the absorbance at 280 nm (thus indicating the presence of proteins) and then flowed into the ICP-MS nebulizer for metal analysis. Four main peaks were observed with elution times ranging from 10-16.5 minutes which we identified as iron protein and iron chelate complexes (vide infra and detailed in Table 1). Once we 13 ACS Paragon Plus Environment


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had verified that we could separate the native plasma iron species, the plasma was doped with sodium ferric gluconate (DBI, SFG, 0.3 - 50 ppm), and applied to the LC-ICP-MS to determine if the SFG drug could be separated from these native species. SFG eluted at 9 minutes, preceding all of the native species providing evidence that the LC-ICP-MS approach allows for the separation of SFG drug from the native iron species present in plasma. In the context of separating nanoparticles via LC-ICP-MS, there is a growing body of literature in which this analytical technique has been applied (e.g. for silver and gold nanoparticles).

44-45

However, to our knowledge, these methods have not been developed and fully

method validated in biologicially relevant matrices (i.e. blood plasma) and the chromatography approaches did not typically use sample preparations that would allow for plasma proteins to retain their native folding and metal loading.

44-45

Our work builds upon this fundamental research on

nanoparticle separation, for application to clinical samples. Specifically, we demonstrate that the iron nanoparticle, SFG, can be separable in plasma from native iron species (iron proteins and small molecule iron complexes). This separation allows for the quantitation of native iron concentration for each iron species in plasma and the quantitation of the iron that is bound to the nanoparticle drug.


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Table 1. Iron Speciation in human plasma doped with SFG. Species

Molecular Mass (kDa)

Native Concentration in plasma

Peak Elution (min)

t (min)

Transferrin

79.7

1.8 – 3.7 g/L

11.5 – 13.5

12.9

Ferritin

450

10 – 10.5

10.5

Albumin

66

10.5 – 11.5

11.4

Citrate

0.43

15 – 16.5

16.4

SFG

289 – 440

9 – 10

10.2

a

a

10 – 250 µg/L

a

a

36.1 – 53.6 g/L

a

0.1 mM

b

c

b

–

a

R

(a) ref 36. (b) ref 27. (c) ref 46.

Figure 3. Diagram of the liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) instrumental setup. The LC setup includes a UV-Vis multiple wavelength detector.

Identification of iron species separated by LC. To identify the peaks that we had separated via LC-ICP-MS, we first estimated their molecular weights by generating a calibration curve with 15 ACS Paragon Plus Environment


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known protein standards: thyroglobulin, ɣ-globulis, albumin, ribonuclease A and paraaminobenzoic acid standards (molecular weights range from 670 to 0.14 kDa). A solution of the standards was injected on to the LC-ICP-MS under the same conditions utilized to separate the species present in plasma, and the elution times for each standard were recorded. As the likely iron species present in plasma are transferrin, albumin and iron citrate, these proteins or complexes were individually spiked into plasma along with the iron nanoparticle drug, sodium ferric gluconate, and the LC-ICP-MS was run. These experiments allowed us to identify the time at which each iron protein or iron species eluted. A shown in Table, 1,

27, 36, 46

the first peak was Sodium

ferric gluconate, SFG, (DBI) (9 – 10 min), followed by ferritin (10 – 10.5 min), albumin (10.5 – 11.5 min), transferrin (TBI) (11.5 – 13.5 min) and finally a iron species that contained iron-citrate (LI) (15 – 16.5 min). To independently confirm our peak identification, the peaks were collected, and their molecular weights were confirmed by MALDI-MS or ESI-MS (Table S1).

Of particular note is our ability to separate and quantify drug bound iron, DBI. Ideally, in pharmacokinetic studies, the breakdown of the active pharmaceutical ingredient (API) is monitored; however, to date for iron nanoparticle drugs, these measures have been indirect as a direct measure was not known. We envision that this ability to measure DBI directly will aid both pharmaceutical companies that are developing new iron nanoparticle products and regulatory agencies in the approval of new iron nanoparticle products.

We also sought to identify the exact species present in the ‘labile iron’ peak. Although the presence of labile iron (LI) is known, its chemical identity and speciation are unresolved. Most previous methods to capture LI have relied on chelators which both displace the iron from its native ligand 16 ACS Paragon Plus Environment

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and capture iron from proteins (other than transferrin) that are present in plasma and therefore do 28

not distinguish or identify LI.

28, 47

We collected the LI peak that we observed after performing SEC

on plasma of a healthy volunteer (between 16 to 16.5 min) and analyzed it by by electrospray ionization (ESI) mass spectrometry.

27, 48-49

The predominant species that we identified by ESI-MS

were iron-citrate complexes: [Fe (cit) H] (m/z 366.4); [Fe (cit) H] (m/z 488.9); [Fe (cit) H ] (m/z 2-

3

-

3

2

2

-

3

3

2

733.8) (Figure 4). Citrate is a known small molecule present in plasma at high concentrations (~ 0.1 mM), therefore the observation that LI includes iron citrate species is consistent with its abundance in plasma. Further evidence for citrate is found in an additional prominent MS peak at 27

m/z 191.0 which corresponds to free citric acid (H cit). This peak is likely native non-iron bound -

3

citrate present in the plasma and/or Fe-citrate that has been dissociated under the mass spectrometry conditions. All of the iron citrate species identified match those reported in previous in vitro studies of iron citrate at neutral pH.

27, 49

These ESI-MS data provide direct evidence that iron citrate is a component of LI in the plasma that we examined. We note that there are other species present in the ESI spectrum, suggesting that LI is made up of a number of low molecular weight iron complexes, and that LI is composed of multiple iron chelate complexes that are in equilibrium. This finding is supported by recent work of Lindahl et al., who separated and sought to identify LI in a series of plasma samples (human, pig, horse, mouse). In their work, they also found multiple species for labile iron, and, intrerestingly, did not find iron citrate as a major species. This may be due to different 35

experimental approaches. Lindahl. et al separated and concentrated the low molecular weight iron species fraction of the plasma prior to application to LC-ICP-MS, performed studies at pH 8.5, 6.5 and 4.5, and identified the iron citrate peaks in the LC by spiking samples with pre-formed iron



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citrate complexes. In contrast, we directly injected the undiluted plasma, performed experiments 35

at pH 7.4 and collected identified iron citrate by collecting labile iron peaks from our described LC separation and directly measuring the accurate mass via ESI-MS. Taken together, these findings reveal that the nature of the LI species is complex and its speciation is affected by experimental conditions.

Figure 4. Averaged electrospray mass spectra of A) iron citrate standard solution at 500 ppm in 10 mM ammonium acetate; B) plasma fraction collected between 16-16.5 min in 10 mM ammonium acetate; C) 10 mM ammonium acetate (blank). All data were acquired on Waters Synapt G2S QTOF mass spectrometer through direct infusion at 10 µL/min in negative ion mode, source temperature 50 °C, capillary voltage 1.5 kV, acquisition time: 1 min. 18 ACS Paragon Plus Environment

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Method Validation. The second goal of this work was to validate our LC-ICP-MS method, per FDA guidelines, so that it can be applied to clinical samples. To our knowledge, LC-ICP-MS of iron speciation has not previously been validated for this application. To this end, the method was validated for sensitivity, linearity, accuracy, precision, recovery and stability (freeze-thaw, benchtop, autosampler/post-preparative, and long-term storage stability) per the FDA guidance for bioanalytical method validation. SFG spiked in plasma served as the calibrant for DBI and holo41

transferrin in 10 mM Tris buffer was used as the calibrant for TBI, PBI, and LI. A summary of the validated analytical performance is provided in Table 2, and described below.

Table 2. Summary of Analytical Performance Analytical Parameter LLOQ Analytical range Calibration curve r Intra-day %CV Intra-day % accuracy Inter-day %CV Inter-day % accuracy Bench-top stability

DBI 0.3 ppm 0.3 ppm – 50 ppm >0.99 1.8 – 4.7 % 97 – 114 % 2.4 – 13.2 % 92 – 122 % 24 h

Post-preparative stability

@RT (25 C): 26 h @4 C: 5 days

2

a

TBI, PBI, LI 10 ppb 10 ppb – 1400 ppb >0.99 0.8 – 10.2 % 82 – 113 % 1.7 – 12.4 % 85 – 107 % N/A @RT (25 C): 63 h @RT (25 C) (LI): 120 min @4 C (LI): 11 h b

c

d

o

o

o

o

e

e

o

Freeze-thaw stability ≤ 15 % deviation (vs. (3 cycles at -20 C) non-F/T sample) N/A Long-term storage stability 6 months 43 days (-20 C) (a) SFG spiked in plasma served as the calibrant for DBI (b) holo-transferrin in 10 mM Tris buffer was used as the calibrant for TBI, PBI, and LI (c) LI in plasma is not stable at room temperature (25 C), was only stable for 1 h on ice (0 C) and is not stable when subjected to freeze-thaw (3 cycles at -20 C). (d) stability for prepared samples of holo-transferrin in 10 mM Tris buffer (e) stability for prepared samples of LI in plasma c

o

o

o

o

o



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Sensitivity and linearity. Typical regression equations for DBI and TBI/PBI/LI exceeded r > 0.99 2

for all calibration curves. The linear range of quantification for DBI and TBI/PBI/LI encompassed over three orders of magnitude of concentration. Specifically, the linear range for DBI was 0.3 ppm – 50 ppm and the linear range for TBI/PBI/LI was 10 ppb – 1400 ppb. Lower limits of quantification (LLOQs) were the lowest point on the calibration curve that also met accuracy and precision criteria of less than 20% deviation. All other calibration points had accuracy and precision of less than 15% deviation. LLOQ for DBI was 0.3 ppm and LLOQ for TBI/PBI/LI was 10 ppb.

Precision, accuracy and recovery. DBI and TBI/PBI/LI were evaluated for intraday and interday precision and accuracy. For DBI, within-run intraday accuracy ranged between 97-114% and interbatch accuracy was between 92-122%. Intraday precision, as assessed by % CV, for DBI was 1.84.7% whereas interday precision was 2.4-13.2%. TBI/PBI/LI had similar analytical performance where within-run intraday accuracy ranged between 82 – 113% and inter-batch accuracy was between 85-107%. Intraday precision for TBI/PBI/LI was 0.8-10.2% whereas interday precision was 1.7-12.4%. Recovery for DBI, as assessed with SFG as a calibrant, ranged between 83-108% across the linear range. Recovery of TBI/PBI/LI, as assessed with holo-transferrin as calibrant, ranged between 82-113% across the linear range.

Stability. Stability was assessed by determining benchtop, freeze-thaw, autosampler/postpreparative, and long-term storage stability. DBI was stable on the bench-top at 25 C for 24 h and o

displayed ≤15% deviation after being subjected to three cycles of freeze-thaw at -20 C. Posto


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preparative stability in a temperature controlled autosampler was 5 days at 4 C and 26 h at 25 C. o

o

Long-term storage of DBI species indicates DBI is stable at -20 °C for 6 months. LI species were the most sensitive to stability concerns in the measurement of TBI, PBI and LI where LI species have significant stability limitations that warrant specific handling. LI in plasma was not stable on the bench-top at room temperature (25 °C) and was only stable on ice (0 °C) for 1 h. Freeze-thaw should be avoided for samples intended for LI determination because LI was not stable when subjected to three cycles of freeze-thaw at -20 °C. The LI component of plasma is stable for up to only one freeze-and-thaw cycle. Due to these constraints, plasma samples intended for LI determination should be frozen immediately upon collection and then prepared immediately upon thawing. Post-preparative temperature had a significant effect on sample stability of LI. Postpreparative stability of LI in a temperature controlled autosampler at 4 °C was 11 h; whereas, if the autosampler was maintained at 25 °C, post-preparative stability was only 2 h. Post-preparative stability of TBI species was more robust as indicated by post-preparative stability of 63 h at 25 °C. Long-term storage of LI was also limited at 43 days at -20 °C.

Proof of Principle: Application of Validated Bioanalytical Method to A Clinical Trial Sample. The third goal of this work was to obtain proof of principal data that the LC-ICP-MS assay that we had developed and validated can be applied to a clinical trial sample. To this end, we evaluated a sample from an ongoing clinical trial in our laboratory. A healthy volunteer was administered 125 mg of sodium ferric gluconate (SFG) after which the volunteer’s blood was sampled at times 0 and 3 h and evaluated for iron speciation. Figure 5 shows the LC-ICP-MS



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chromatograms that were obtained at 0 and 3 h. The chromatogram colored red shows the volunteer’s native iron distribution prior to SFG IV iron treatment (i.e. T = 0).

Figure 5. LC-ICP-MS Chromatograms of human plasma pre (red) and 3 hours post (blue) infusion with SFG. ICP-MS chromatogram for counts of iron (56). Inset is an expansion of the labile iron peak. * = internal standard

The first peak in the chromatogram (~ 1 min) is the internal standard which is added post column. The next peak in the T=0 h (red), which appears around 11 min, is protein bound iron (PBI, Fealbumin and Fe-ferritin). This is followed by the largest peak at T=0 h, TBI, around 12 min, followed by the least abundant peak LI (Fe-citrate) at 16 min. The chromatogram colored blue shows the volunteer’s plasma 3 h after infusion. Two key features are noted in the T=3 h postinfusion plasma: (1) a new peak appears at 9 - 10 min and is assigned DBI and (2) TBI and LI peaks increase compared to their pre-infusion levels. Quantification of DBI yielded concentrations of 5110 parts per billion (ppb) at 3 h. The increases in TBI and LI measure were 1,678 ppb (1133 ppb at 0 h and 2811 ppb at 3 h) for iron-bound transferrin and 38 ppb (24 ppb at 0 h and 62 ppb at 3 h) for labile iron. Together, these data provide strong evidence that the LC-ICP-MS can be applied to clinical samples of patients administered iron nanoparticle drugs, and that the data 22 ACS Paragon Plus Environment

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obtained includes a measure of the drug bound iron (DBI). These are the first clinical data for which both DBI and LI are directly measured in their native forms. The development and validation of this method to measure iron speciation in clinical samples after the administration of iron-nanoparticles has the potential to be expanded to study other iron products (e.g. there are five other FDA approved IV iron drugs). These other iron products differ with respect to their average molecular weights, iron core sizes, stabilizing carbohydrate ligand(s), and average particle size, and therefore may exhibit different chromotographic properties. Therefore, to expand to these other products exploratory LC work is needed. Ultimately, this method has the potential to inform on the speciation of other metal nanoparticles (e.g. gold nanoparticles), but each individual type of nanoparticle must be tested to determine the suitability of this assay.

CONCLUSIONS Nanomedicines show great promise as next generation therapies; however, their development has been stymied by their molecular complexity which makes it difficult to track and assess activity and potency in biological settings. One of the most clinically successful nanomedicines are the iron carbohydrate colloids for the treatment of iron deficiency anemia (IDA). These drugs serve to release iron in plasma which is then delivered to cells via transferrin and taken up by iron proteins that are iron deplete. IDA is a growing public health concern, as it accompanies diseases that impact large populations including chronic kidney disease (CKD) (30 million Americans affected) 23 ACS Paragon Plus Environment


and cancer (1.7 million Americans predicted to be diagnosed in 2018).

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50-51

There is strong interest

in approving new innovator and generic iron nanoparticle drugs for IDA treatment; however, the lack of tools to monitor the fate of iron nanoparticles in clinical samples has impeded their development. Our novel Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry (LC-ICP-MS) method allows for the first direct quantification of the iron-carbohydrate nanoparticle drug (DBI) in clinical samples, while simultaneously measuring the speciation of the iron released from the nanoparticles. This method offers a much-needed tool to track iron speciation. Moreover, the method provides a snapshot of metal distribution in biological samples and has potential applications in the development of new metal-based nanomedicines and in understanding metal speciation in biological systems.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Data processing, iron species characterization, and method validation is presented in the supporting information.

AUTHOR INFORMATION Corresponding Authors * [email protected]


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ORCID: Heather M. Neu: 0000-0002-8204-7392; Sergei A. Alexishin: 0000-0002-6518-8812; James E. Polli: 0000-0002-5274-4314; Maureen A. Kane: 0000-0002-5525-9170; Sarah L. J. Michel: 00000002-6366-2453 Author contributions. S.L.J.M. conceived of the approach, H.M.N., M.A.K., J.E.P. and S.L.J.M. designed the research. H.M.N., S.A.A., J.E.P.B., W.J. and A.M.C.W. performed research. J.M.F, A.Z. and J.E.P. collected clinical trial sample, D.S., N.Z., and W.J., provided oversight, S.L.J.M., H.M.N., M.A.K, J.E.P., and S.A.A. wrote the manuscript. Disclaimer. This article reflects the views of the author and should not be construed to represent FDA’s views of policies. Competing financial interests. The authors declare no competing financial interests ACKNOWLEDGEMENTS S.L.J.M., M.A.K. and J.E.P are grateful to the FDA for support of this work (UO1FD005266). Additional support from the FDA (1U01FD005946) for H.M.N. and J.E.P. is also gratefully acknowledged. Additional support was also provided by the University of Maryland School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014).



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For Table of Contents Use Only


Snapshots of Iron Speciation: Tracking the Fate of Iron Nanoparticle

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