In vivo Stability and Biodistribution of Superparamagnetic

Iron Oxide Nanoparticles Radiolabeled with Indium-111

Master’s Thesis

By

Haotian Wang

Advisor: Dr. Samuel John Gatley

to

The Bouvé Graduate School of Health Sciences

In Partial Fulfillment of the Requirements for the Degree of Master of

Science in Pharmaceutical Science with Specialization in

Pharmaceutics and Drug Delivery

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

January, 2014

ABSTRACT

Nanoparticles of various kinds have generated considerable excitement as vehicles for drug

delivery, and as imaging agents (Yen, 2013). Magnetic nanoparticles are of particular

interest since their distribution in the body can be manipulated using magnetic fields, and

they can also act as highly sensitive contrast agents in magnetic resonance imaging (MRI)

(Bonnemain, 1998; Wang, Hussain, & Krestin, 2001). For some applications, radionuclide

imaging (Positron emission tomography (PET) or Single-photon emission computed

tomography (SPECT)) offers advantages when used in conjunction with MRI, since the

strengths of the two modalities, higher spatial resolution for MRI, but better quantification

for PET or SPECT, are complementary (Bouziotis et al., 2012). On the other hand, the

validity of dual modality imaging requires that the association between radioactive label

and magnetic label remains intact in the human or animal body during the period of study.

Evaluation of the extent to which this requirement is true for PEGylated nanoparticles

containing a core of superparamagnetic iron oxide (SPION), and also labeled with a

radioactive metal which is commonly used in nuclear medicine, indium-111, is a major

component of this proposal.

In the present study, we prepared SPIONs using co-precipitation of iron oxide,

complexation with oleic acid, and final coating of the particles with DSPE-PEG plus

DMPE-DTPA using the lipid rehydration technique. We radiolabeled the particles by

introducing iron-59 ferric chloride or carbon-14 oleic acid during their preparation, and by

forming a chelate between the anchored DTPA and indium-111. We conducted

biodistribution studies in mice with particles labeled with each radionuclide, and also with

indium-111 labeled indium citrate, indium-DTPA, and DMPE-DTPA-indium, and with

carbon-14 oleic acid. We found that that the radioactivity from radioindium labeled

SPIONs was, as hypothesized, preferentially localized in liver, spleen and bone associated

with trapping of colloids by reticuloendothelial cells. However, radioactivity from SPIONs

labeled with iron-59 was accumulated in liver, spleen and bone to a greater extent than

from indium-111 labeled SPIONs, showing that the association of indium with the particles

is not completely maintained in vivo. Radioactivity from SPIONs labeled with carbon-

14 oleic acid exhibited a quite different distribution pattern to iron-59 or indium-111, and

so does not remain with the iron oxide core after injection into mice.

NORTHEASTERN UNIVERSITY

Graduate School of Bouvé College of Health Sciences

Thesis Approval

Thesis title: In vivo Stability and Biodistribution of Superparamagnetic Iron Oxide

Nanoparticles Radiolabeled with Indium-111

Author: Haotian Wang

Program: Pharmaceutical Sciences

Approval for thesis requirements for the Master of Science Degree

in Pharmaceutical Sciences

Thesis Committee

(Chairman) Date

Other committee members:

Date

Date

Date

Dean of the Bouvé College Graduate School of Health Sciences:

Date

5

ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude and deepest appreciation to my supervisor,

Dr. John Gatley, for his invaluable advice and support throughout my project. His

brilliant ideas, guidance and mentoring helped me overcome many hurdles throughout

my Master’s research. Without your understanding and patience none of this would

have been possible.

I would like to thank my thesis committee members, Dr. Richard Duclos, Dr. Rajiv

Kumar and Dr. Ban-an Khaw, for their time, invaluable advice on my thesis project and

moral support.

I would particularly like to thank Dr. Dattatri Nagesha for his help and support in the

initial setting up of the project and for guiding me throughout.

Special thanks to Dr. Srinivas Sridhar for allowing me to learn the synthesis of SPIONs.

My sincere thanks to Sarom, Kun, Shilpa, Nidhi, Yu, Codi, Jodi, Rita and all my friends

who have given their time, their knowledge and their resources to help me.

Last but most importantly, I want to thank my parents, who have sacrificed much to

ensure that I have the best in life. And finally, I would like to thank my wonderful wife,

Lu, who has been there for me through the toughest of times and completely supporting

my dreams.

6

TABLE OF CONTENTS

ABSTRACT .................................................................................................................. 2

ACKNOWLEDGEMENTS ........................................................................................ 5

TABLE OF CONTENTS ............................................................................................. 6

LIST OF TABLES ..................................................................................................... 9

LIST OF FIGURES ................................................................................................. 10

1. INTRODUCTION.................................................................................................. 12

1.1 Brief Introduction to the History of Nanotechnology ....................................... 12

1.2 Nanoparticles Platform ...................................................................................... 15

1.2.1 Liposomes ............................................................................................... 15

1.2.2 Polymeric micelles .................................................................................. 16

1.2.3 Superparamagnetic Iron Oxide Nanoparticles (SPIONs) ....................... 17

1.3 Rationale for each Specific Aim ....................................................................... 18

2. SPECIFIC AIMS .................................................................................................... 22

2.1 Objectives .......................................................................................................... 22

2.2 Hypothesis ......................................................................................................... 23

7

3. MATERIAL AND METHODS ............................................................................. 25

3.1 Animals ............................................................................................................. 25

3.2 Chemical Reagents ............................................................................................ 25

3.3 Preparation of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) .......... 26

3.4 Preparation of DSPE-PEGylated SPIONs ........................................................ 27

3.5 Characterization of PEGylated SPIONs ............................................................ 27

3.5.1 Particle Size and Surface Charge Analysis ............................................. 27

3.5.2 Transmission Electron Microscopy (TEM) ............................................ 28

3.6 Radiolabeling PEGylated SPIONs with Indium-111 ........................................ 28

3.7 Biodistribution Studies of indium-111 Radiolabeled PEGylated SPIONs........ 29

3.8 Biodistribution Studies of indium-111 Radiolabeled indium citrate, indium-

DTPA and DMPE-DTPA-indium ............................................................................ 29

3.8.1 Preparation of indium-111 radiolabeled indium citrate solution ............ 29

3.8.2 Preparation of indium-111 radiolabeled indium-DTPA solution ............ 30

3.8.3 Preparation of indium-111 radiolabeled DMPE-DTPA-indium solution 30

3.8.4 In vivo Biodistribution Studies................................................................ 30

8

3.9 Biodistribution studies of iron-59 and carbon-14 oleic acid radiolabeled

PEGylated SPIONs ................................................................................................. 31

3.9.1 Preparation of iron-59 or carbon-14 oleic acid radiolabeled SPIONs .... 31

3.9.2 In vivo biodistribution studies ................................................................. 32

4. RESULTS AND DISCUSSIONS........................................................................... 33

4.1 Preparation and Characterization of PEGylated SPIONs ................................. 33

4.2 Radiolabeling of PEGylated SPIONs with Indium-111 .................................... 35

4.3 Biodistribution of indium-111 radiolabeled PEGylated SPIONs ...................... 35

4.4 Biodistribution of indium-111 radiolabeled indium citrate, indium-DTPA and

DMPE-DTPA-indium .............................................................................................. 38

4.5 Biodistribution of iron-59 or carbon-14 oleic acid radiolabeled PEGylated

SPIONs .................................................................................................................... 43

4.6 OVERALL CONCLUSIONS ........................................................................... 49

5. LIMITATIONS AND FUTURE DIRECTIONS ............................................... 51

6. BIBLIOGRAPHY ............................................................................................... 52

9

LIST OF TABLES

Page Number

Table 1: Radioactivity recovered of 111indium with SPIONs in 35

successive filtration.

Table 2: Biodistribution data of indium-111 labeled PEGylated SPIONs 36

at different time points.

Table 3: Biodistribution data of indium-111 labeled DMPE-DTPA- 42

indium, indium citrate and indium-DTPA.

Table 4: Biodistribution data of iron-59 labeled PEGylated SPIONs 44

Table 5: Biodistribution data of carbon-14 oleic acid labeled PEGylated 45

SPIONs.

10

LIST OF FIGURES

Page Number

Figure 1: Photographs of (a) the Lycurgus cup; and (b) Church 14

window in the Cathédrale Notre-Dame de Chartres.

Figure 2: Schematic structure of (a) non-modified liposomes; 16

(b) PEGylated liposomes with PEG coated on the surface;

(c) immune-liposomes with antibodies attached to the

surface.

Figure 3: Schematic structure of self-assembled micelles. 17

Figure 4: Schematic diagram of oleic acid stabilized SPIONs. 22

Figure 5: Schematic structure of water dispersible polyethylene glycol 23

SPIONs.

Figure 6: Particles size of PEGylated SPIONs measured using 33

dynamic light scattering in 90Plus particle size analyzer

Figure 7: Stability Measurement of PEGylated SPIONs for consistent 34

7 days.

Figure 8: TEM micrograph of the iron core of PEGylated SPIONs 34

Figure 9: Graph of biodistribution of 111indium labeled PEGylated 37

SPIONs at different time points.

Figure 10: Indium-111 in urine after administration of 111indium 37

labeled PEGylated SPIONs.

Figure 11: Graphs showing liver-to-blood (left) and brain-to-blood 38

(right) ratios versus time.

11

Page Number

Figure 12: Comparison of biodistribution between indium-SPIONs 39

and DMPE-DTPA-indium at 30 min and 2 h.

Figure 13: Comparison of liver-to-blood ratios versus time between. 39

indium-SPIONs and DMPE-DTPA-indium.

Figure 14: Comparison of urine radioactivity between indium-SPIONs 40

and DMPE-DTPA-indium.

Figure 15: Comparison of biodistribution of indium-SPIONs, DMPE- 42

DTPA-indium and indium citrate at 30 minutes post

injection.

Figure 16: Comparison of indium-111, iron-59, and carbon-14 oleic 46

acid biodistribution patterns in liver, spleen, bone, blood

and kidney at 10 min, 30 min, and 24 h time points.

12

1. INTRODUCTION

1.1 Brief Introduction to the History of Nanotechnology

According to the International Union of Pure and Applied Chemistry (IUPAC),

International Organization for Standardization (ISO), American Society of Testing and

Materials (ASTM) and National Institute of Occupational Safety and Health (NIOSH),

nanoparticles are defined as particles of size between 1 and 100 nm (Vert et al., 2012;

Horikoshi & Serpone, 2013). Although terms such as “nanoparticle” and

“nanotechnology”, which was first coined by Professor Taniguchi (1974), are of fairly

modern origin practical uses of nanoparticles predate recognition of their existence by

many centuries (Colomban, 2009). In ancient Rome (4th Century A.D.) craftsmen knew

how to produce colored glasses, sculptures and paintings that we now know owed their

optical properties to gold and silver nanoparticles (Hougha, Noblea, & Reich, 2011).

The Lycurgus Cup (Figure 1a) in the British Museum of London is an amazing example.

Its color changes from green in daylight to red when illuminated from the inside

(Heiligtag & Niederberger, 2013; Freestone et al., 2007). From the Medieval Period to

the Renaissance Period, stained glass church windows (Figure 1b) of breathtaking

beauty were made that owed their ruby red color to gold nanoparticles and their deep

yellow color to silver nanoparticles (Jin et al., 2001) and the size of metal nanoparticles

is critical to producing particular colors. The astonishing discovery in nanoparticle

research was made by Michael Faraday approximately 157 years ago (1857). Faraday

prepared his deep red gold dispersions by reduction of an aqueous solution of

13

chloroaurate (Na[AuCl4]) by phosphorus in carbon disulfide (a two-phase system). The

red “solution” was due to the optical property of gold nanoparticles. Later, such

“solutions” became known as colloidal systems, which are defined as particles of size

between 1 and 1000 nm of one material or composition suspended in another material.

Milk, blood and paint are colloidal systems. In fact, Faraday’s systematic studies on the

interaction between light and metal nanoparticles is considered as the threshold of

modern colloid chemistry and the beginning of Nanoscience and Nanotechnology

(Edwards & Thomas, 2007). Thus there is considerable overlap in terminology between

“nanoparticles” and “colloids”. An early medical application of a colloid was the X-ray

contrast agent Thorotrast (Dickson 1932). This consisted of an injected preparation of

particles of thorium-232 dioxide that were taken up by reticuloendothelial cells in the

liver and, since thorium is opaque to X-rays, permitted medical imaging of this organ.

It was used worldwide from 1930 to 1950 (Kaick. Et al., 1999; Jellinek, 2004).

Unfortunately, thorium dioxide particles remained in the liver for the patients’ lifetime

and is radioactive, and since thorium emits alpha particles, many people developed liver

cancer many years later. This may be the first medical use of a nanoparticle. Another

imaging application example is the utilization of sulfur colloid, labeled with

Technetium-99m for liver and spleen scanning (Stern, McAfee, & Subramanian, 1966).

This radiopharmaceutical is still used today in nuclear medicine (Bhalani et al., 2012).

14

Figure 1. Photographs of (a) the Lycurgus cup displays different colors depending on

whether it is illuminated externally (left) or internally (right) (The British Museum); (b)

Church window in the Cathédrale Notre-Dame de Chartres (France). (Horikoshi &

Serpone, 2013)

In the 20th century, a much better understanding of colloidal systems developed due to

advances in physical instrumentation. For example, conventional light microscopes are

unable to visualize objects as small as 100 nm, but the development of the electron

microscope by German scientists Max Knoll and Ernst Ruska in 1931 enabled

researchers to detect and measure objects in this size-range.

Additionally, in the early 20th century, the idea of targeted delivery to enhance drug

therapy was conceptualized by German scientist Dr. Paul Ehrlich. The word

“Zauberkugeln” – “Magic Bullets” – in English was coined to describe selectively

targeted chemotherapy (Kreuter, 2007; Yordanov, 2013). Ehrlich’s notion was the use

of histological dyes, which had affinity for syphilis bacteria in tissues, to deliver arsenic,

which was toxic to the bacteria. The dye was the vehicle and arsenic was the cargo

(Ehrlich, 1877). Salvarsan was actually a low molecular weight compound, but the idea

of drug delivery vehicles is now more commonly associated with nanoparticles.

15

Nowadays, the literature on this subject is very large. A MEDLINE search on

“nanoparticle” and “drug delivery” on January 9th, 2014 yielded almost 17,000 articles.

1.2 Nanoparticles Platform

Nanotechnologies have the potential to revolutionize the drug development process and

change the landscape of the pharmaceutical industry (Allen & Cullis, 2004; Shi et al.,

2010). Many different nanoparticles platforms have shown promise in delivering drugs

to desired tissue sites in the body such as liposomes, polymeric micelles, dendrimers,

etc. (Farokhzad & Langer, 2009) Because these carriers have high loading efficiencies

and protect drugs from undesired in vivo interference, nanotechnologies can improve

the therapeutic effect of drugs by enhancing their efficacy and/or increasing their

tolerability in the body (Swami et al., 2010; Zhang et al., 2007). Here are some

examples:

1.2.1 Liposomes

Liposomes are multilayer vesicular platform made with interior aqueous phospholipid

(such as phosphatidylcholine) bilayered membrane structures (Antimisiaris, Kallinteri,

& Fatouros, 2007). Hydrophobic agents can be incorporated into the phospholipid

membrane, while hydrophilic agents can be inserted into the aqueous interior. The

vesicles size can range from several microns to 30 nm (Barenholz, 2001). The

encapsulated drugs can be released in the body when the liposomes are broken down

by enzymes or by the fusion of the liposomal bilayer with the cell membrane or

16

endosomal membrane (Farokhzad & Langer, 2006). Moreover, liposomes have

properties including long circulation time in the body system, ease of surface

modification, and fewer side effects that make them well suited as drug delivery carriers

(Antimisiaris, Kallinteri, & Fatouros, 2007). Current research is focused on the

development of multifunctional nanocarriers that combine the advantages of the basic

types leading to improved efficacy (Torchilin, 2009). Major types of liposomes are

shown in Figure 2. Liposomes were the first nano-based drug carriers to be approved

by the Food and Drug Administration (FDA) for clinical use. For instance, DOXIL®

(doxorubicin liposomes) was approved in 1995 for the treatment of patients with

ovarian cancer whose disease has progressed or recurred after platinum-based

chemotherapy (Barenholz, 2012).

Figure 2. Schematic structure of (a) non-modified liposomes; (b) PEGylated liposomes

with PEG coated on the surface make liposomes more stable and long circulating; (c)

immune-liposomes with antibodies attached to the surface intended for recognizing

specific target receptors and antigens. (Yordanov, 2013)

1.2.2 Polymeric micelles

Polymeric micelles (Figure 3) are spontaneously self-assembling block copolymers

with an inner hydrophobic core and hydrophilic shell, such as PEG, in aqueous solution

at concentration above the critical micellar concentration (CMC) (Chan et al., 2010).

17

Polymeric micelles have proved that they have great potential modalities as therapeutic

drug carriers (Matsumura & Kataoka, 2009; Nishiyama & Kataoka, 2006). As drug

delivery carriers, polymeric micelles exhibit several unique properties including ease

of preparation, long circulation in bloodstream, reduced adverse effects, ultra small

particles size, as well as reaching target sites and controlled drug release, thus making

them promising for effective tumor accumulation of therapeutic agents by the EPR-

based mechanism (Bae et al., 2005; Nishiyama et al., 2005).

Figure 3. Schematic structure of self-assembled micelles (Yordanov, 2013).

1.2.3 Superparamagnetic Iron Oxide Nanoparticles (SPIONs)

Superparamagnetic iron oxide nanoparticles, magnetite (Fe3O4) and maghemite (γ-

Fe2O3) with a small particle size of 5-25 nm, with appropriate surface coatings have

higher capabilities as MRI contrast enhancers than conventional contrast agents such

as gadolinium chelates (Shanehsazzadeh et al., 2013; Thomas, Park, & Jeong, 2013;

Chen et al., 2010). SPIONs also have potential applications in drug delivery and in

detoxification of biological fluids (Neuberger et al., 2005; Lodhia et al., 2010). The

essential requirement for the use of SPIONs in these applications is a controlled particle

size smaller than 100 nm with a narrow distribution and high magnetisation value

18

because the properties of the nanocrystals depend on their size and superparamagnetism

(Gupta & Gupta, 2005). Uncoated iron nanoparticles tend to form large aggregates.

Degradable nontoxic and biocompatible polymers such as PEG, poly(lactide-co-

glycolide) (PLGA), and dextran are used to coat the iron oxide nanoparticles to prevent

aggregation (Teja & Koh, 2009). Surfactants and surface coatings used can affect size,

surface charge and physiological stability of nanoparticles, so that different

pharmacokinetics and biodistributions can be obtained. A potential advantage of

SPIONs is that they can be attracted to a tumor or organ by using an external magnet

(Gupta and Gupta, 2005; Sawant, 2008).

Among all the techniques to synthesize SPIONs, the co-precipitation method is the

easiest and cheapest (Qu et al., 1999; Gupta & Wells, 2004). The method has many

advantages: the production scale can be easily amplified or reduced, and the particle

size distribution ranges from 5 to 25 nm (Gupta and Curtis, 2004).

1.3 Rationale for each Specific Aim

Rationale for Aim 1: Expertize in making and characterizing this type of nanoparticle

is available in the laboratory of Dr. Sridhar, which will greatly facilitate its preparation

in our own hands. Dr. Sridhar’s group also has experience in using this kind of particles

in MRI experiments. It will be necessary to transfer the methodology to our own

laboratory, in order to work with radioactive materials.

19

Rationale for Aim 2: This method of radiolabeling has been used extensively with

nanoparticles such as liposomes. The phospholipid hydrocarbon tails incorporate

themselves into the lipid membrane, while the polar ethanolamine-phosphate head

group is exposed on the surface of the particles. DTPA attached to the ethanolamine

moiety via an amide bond chelates trivalent metal cations with very high affinity. One

such metal is indium, and a radioisotope of this metal, indium-111, is commonly used

in clinical nuclear medicine. It emits gamma-rays of energy 0.17 and 0.25 MeV, which

combine the features of good ability to penetrate the human body, with efficient

detection using nuclear medicine imaging instruments. The half-life is 2.8 days, which

offers a reasonable “shelf-life” in the radiopharmacy with acceptably rapid clearance

from the patient by physical decay after imaging studies have been completed. The

electron-capture mode of decay avoids the higher radiation doses to tissues associated

with nuclides that emit beta particles.

Rationale for Aim 3: Mice were chosen as test subjects for tissue distribution

experiments following long established practice in experimental nuclear medicine. As

fellow mammals, they are similar in terms of basic biochemistry and physiology to

humans, and candidate radiopharmaceuticals can be conveniently administered via one

of the prominent tail veins. Particles of sub-micron to micron size-range are well known

to be accumulated from the blood by reticuloendothelial cells in the liver and spleen,

and therefore if the radio-indium remains associated with the nanoparticles one should

find the radioactivity predominantly in these organs following intravenous injection.

20

Rationale for Aim 4: Since indium is attached to DTPA via chelation, the DTPA is

itself attached to phosphatidyl ethanolamine via an amide bond, and the DMPE-DTPA

is attached to the nanoparticles via hydrophobic forces, it seems possible that any of

these labeled species (indium citrate, indium-DTPA or DMPE-DTPA-indium) could be

released from the particles. However, they are all expected to have biodistribution

patterns that are distinct from that of indium labeled PEGylated SPIONs and distinct

also from each other. A finding that none of these species of indium-111 (indium citrate,

indium-DTPA or DMPE-DTPA-indium) selectively localizes in liver or spleen (as

hypothesized for indium-111 administered as labeled nanoparticles) would therefore

strengthen the argument that indium-111 remains firmly associated with the SPIONs in

vivo.

One possible complication when using indium3+ is the formation of a colloidal form of

indium oxide that can also be accumulated by endothelial cells (Ebbe et al., 1996). For

this reason, indium-111 is handled in dilute citric acid which forms a weak chelate

compound, indium citrate. In the circulation, indium is strongly chelated by transferrin.

A complication in administering the modified phospholipid DMPE-DTPA-indium is

the possible formation of micelles, which could also be cleared by reticuloendothelial

cells.

21

Rationale for Aim 5: If the nanoparticles remain intact after in the blood after

administration, then all the components, iron oxide core, intermediate oleic acid layer

and DMPE-DTPA-indium on the surface should be delivered to various organs to the

same degree. If the core is labeled with a radioisotope of iron, and the oleic acid is

labeled with a radioisotope of carbon, then the organ distributions of these nuclides

after intravenous administration should be the same as that seen with indium-111

labeled nanoparticles. However, while our studies were in progress, Freund et al. (2012)

reported that carbon-14 oleic acid label may be rapidly removed from the iron core after

intravenous injection of similarly labeled SPIONs, so that carbon-14 labeled particles

were unsuitable for in vivo studies. We sought to confirm or refute the findings of

Freund et al (2012).

22

2. SPECIFIC AIMS

2.1 Objectives

1. Prepare and characterize SPIONs with an iron oxide core, an intermediate layer (the

surfactant) of oleic acid and a surface coating of distearoyl phosphatidyl

ethanolamine polyethylene glycol (DSPE-PEG) (Figure 4 and 5).

Figure 4. Schematic diagram of oleic acid stabilized superparamagnetic iron oxide

nanoparticles (Herranz, Pellico, & Ruiz-Cabello, 2012). As detailed in section 3.3, SPIONs

nanoparticles are precipitated from a mixture of ferrous chloride and ferric chloride by

addition of ammonium hydroxide. When oleic acid is added to the mixture, it induces the

formation of oleic acid coated particles that can be handled as a dispersion in chloroform

(Gupta & Wells, 2004).

2. Evaluate the labeling of the SPIONs with radionuclide indium-111 by means of a

lipid anchored chelating group of dimyristoyl phosphatidyl ethanolamine

diethylenetriaminepentaacetic acid (DMPE-DTPA) (Figure 5).

3. Measure the concentration of indium-111 in major organs as a function of time after

intravenous administration of indium-111 labeled SPIONs.

4. Compare the disposition of indium-111 from labeled SPIONs with the disposition

of indium citrate, indium-DTPA and DMPE-DTPA-indium.

23

5. Compare in vivo disposition of indium-111 labeled SPIONs with the dispositions

of iron-59 and carbon-14 oleic acid labeled SPIONs.

Figure 5. Schematic structure of water dispersible polyethylene glycol SPIONs formed by

hydrophobic interaction between lipids. As detailed in section 3.4, addition of DSPE-

mPEG(2000) to oleic acid coated SPIONs results in PEGylated particles that can be

handled in aqueous media. (Yang et al., 2009)

2.2 Hypothesis

1. Indium-111 will become firmly attached to nanoparticles into whose surface coating

DMPE-DTPA has been incorporated, as shown by recovery of indium-111 with

nanoparticles when these are harvested by ultrafiltration.

2. Radioactivity will become relatively concentrated in liver and spleen at early times

after intravenous administration because of trapping of particles by

reticuloendothelial cells, and will remain located in these organs for many hours.

3. Following intravenous administration of indium-111 in the chemical forms of

indium citrate, indium-DTPA or DMPE-DTPA-indium, the radioactivity

concentrations in liver and spleen will be significantly lower than the liver and

spleen radioactivity concentrations measured after administration of indium-111

labeled SPIONs.

24

4. Disposition patterns of radioactivity from nanoparticles labeled with iron-59 in the

core, with carbon-14 in the oleic acid layer, or with indium-111 on the surface will

be different because a very recent article comparing iron-59 and carbon-14 oleic

acid labeling of similarly prepared particles reports that the carbon-14 label may

not be stable in vivo (Freund et al., 2012).

25

3. MATERIAL AND METHODS

3.1 Animals

Male Swiss Webster mice (Charles River laboratories, Cambridge, MA) weighed 25 ~

30 g were used for all in vivo studies. Mice are maintained at the animal facility of

Division of Laboratory Animal Medicine (DLAM) on 12 hour alternating light and dark

period, with access to food and water ad libitum. Mice were treated in compliance with

NIH guidelines for the use of laboratory animals and according to a protocol approved

by Northeastern University Institutional Animal Care and Use Committee (IACUC).

3.2 Chemical Reagents

Iron (II) chloride tetrahydrate (99+%, Acros Organics) (FeCl2.4H2O), Iron (III) chloride

hexahydrate (99+%, Acros Organics) (FeCl3.6H2O) and Ethanol (200 proof, USP/NF)

were purchased from Fisher Scientific (NJ). Sodium Chloride (ACS Reagent, 99+%),

Oleic acid (99+%), Ammonium hydroxide solution (ACS reagent, 28.0-30.0% NH3

basis), Chloroform (ACS Reagent, 99.8+%), and HPLC grade water were purchased

from Sigma-Aldrich (Saint Louis, MO). 1,2-distearoyl-sn-glycero-3-

phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-

mPEG2000) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-

diethylenetriaminepentaacetic acid (DMPE-DTPA) were purchased from Avanti Polar

Lipids (Alabaster, AL). Indium-111 Radionuclide indium citrate, Iron-59 Radionuclide

ferric chloride, [1-14C]-Oleic Acid, SolvableTM and Ultima Gold™ XR were purchased

26

from Perkin Elmer (Waltham, MA).

3.3 Preparation of Superparamagnetic Iron Oxide Nanoparticles (SPIONs)

The SPIONs were prepared by using the co-precipitation method with slight

modifications. Briefly, 15 mL of 0.1 M FeCl2.4H2O with 30 mL of 0.1 M FeCl3.6H2O

were mixed in a round bottom flask equipped with a temperature probe. The molar ratio

of Fe2+ and Fe3+ needs to be 1:2 (Schwertmann & Cornell, 1991). The solution was

bubbled with argon and stirred for 20 minutes in the chemical hood before heating.

When the temperature reached about 80 °C, 3 mL of 5 M NH4OH was added drop-wise,

the clear pale yellow-green solution immediately turned dark brown-black indicating

the formation of iron oxide nanoparticles. At that point, 100 mg (111.7 μL) of oleic acid

was added into the mixture. The heating continued for another 30 minutes while the

temperature was maintained at 80 °C. The sample was then allowed to cool to room

temperature (RT). The resulting SPIONs were separated from the solution using a

strong magnet and the particles were washed with ethanol twice, dried under argon, and

re-dispersed in chloroform. Using a 10 mL syringe (Fisher Scientific, NJ), the re-

dispersed SPIONs were filtered through PVDF 0.45 μm filters (Millipore™ Millex™

Sterile Syringe Filters, Fisher Scientific, NJ) to remove large aggregates, dried under

argon, carefully weighed and re-dispersed in chloroform to produce a 20 mg/mL

SPIONs colloidal solution.

27

3.4 Preparation of DSPE-PEGylated SPIONs

PEGylated SPIONs were prepared by the rehydration method. In brief, 10 mg of DSPE-

mPEG2000 and 2.5 mg of SPIONs were mixed in chloroform. The organic solvent was

removed by using a rotary evaporator to complete dryness. The film was then warmed

in 80 °C water for 1 minute and rehydrated using 1 mL of HPLC grade water. The film

was placed in a sonicating bath for 20 minutes to obtain PEGylated SPION. Uncoated

particles were removed by applying an external magnet, and the supernatant was

removed to a fresh vial.

3.5 Characterization of PEGylated SPIONs

3.5.1 Particle Size and Surface Charge Analysis

The particle size distribution and surface charge (zeta potential) of the PEGylated

SPIONs were determined by dynamic light scattering (DLS) measurement with

Brookhaven Instrument’s 90Plus particle size analyzer (Holtsville, NY). The particle

size was determined at 90° angle and at 25 °C temperature and the average count rate

was adjusted in the range of 100 - 500 kcps by proper dilution (10 μL of the PEGylated

SPIONs solution was diluted in 2 mL deionized water). The mean number diameter of

the particles was obtained on MSD distribution mode. The average zeta potential values

were determined based on the electrophoretic mobility using the Smoluchowski-

Helmholtz equation (Cho, Lee, & Frey, 2012). Each sample was performed five times

with 10 cycles each time and an average value was obtained from the five

28

measurements. The PEGylated SPIONs were stored at 4 °C and their stability was

assessed on day 2, day 3, day 4, day 5, day 6 and day 7.

3.5.2 Transmission Electron Microscopy (TEM)

A JEOL 100-X transmission electron microscope (Peabody, MA) was utilized for

analyzing the structure of the iron core of the PEGylated SPIONs. 10 μL of the

PEGylated SPIONs solution was properly diluted in HPLC grade water and placed on

the carbon-coated copper grids and allowed to air-dry at RT.

3.6 Radiolabeling PEGylated SPIONs with Indium-111

To radiolabel PEGylated SPIONs with indium-111, the particles were synthesized as

described above along with addition of 0.5 mol% of DMPE-DTPA to the lipid film.

This was about 0.02 mg (Sawant, 2008). 1 mL of DMPE-DTPA containing SPIONs

were incubated for 1 hour with 10 μL of 111indium citrate solution (diluted to

approximately 8 - 10 μCi by using 0.5 M HCl) at RT, to allow for the transchelation of

indium-111 from a weak citrate complex into a strong DTPA complex. The radioactivity

was measured in an ionization chamber (CAL/RAD MARK IV, Fluke Biomedical) with

a setting of 676. The unbound indium-111 was removed by a 30 Kilo Dalton cut

microcentrifuge filter (Amicon Ultra 0.5 mL centrifugal filters, Sigma-Aldrich, St.

Louis, MO) at 14,000 rpm for 15 minutes at 4 °C for 3 times. The radiolabeled

PEGylated SPIONs were recovered from the filter using 1.5 mL of 0.9% NaCl solutions

(saline).

29

3.7 Biodistribution Studies of indium-111 Radiolabeled PEGylated SPIONs

Indium-111 labeled PEGylated SPIONs were administered to Male Swiss Webster mice

(25-30 g) via a tail vein. 0.2 mL of indium-111 labeled SPIONs saline solution

(containing about 0.3 mg SPIONs and 1 µCi radioactivity) was injected into each mouse.

The mice were euthanized by cervical dislocation at times 10 minutes, 30 minutes, 1

hour, 2 hours and 24 hours post injection. Five mice were used for each time point.

After euthanasia, blood, urine and other solid organs (brain, heart, liver, lung, spleen,

kidney, testis, fat, bone, muscle and skin) were collected. Each sample was carefully

weighed and placed in the bottom 1 cm of a 12 × 75 mm tube and counted for indium-

111 radioactivity as count per minute (CPM) using a Cobra auto-gamma counter

(Packard, a Canberra company). Lower and upper window thresholds in the counting

protocol were 100 and 300 keV, respectively. From the data, the percentage of injected

dose of radioactivity per gram (ID/g %) of each sample was calculated by 100 × (Tissue

CPM – Background CPM) × Weight (g) / (Injected CPM – Background CPM).

3.8 Biodistribution Studies of indium-111 Radiolabeled indium citrate, indium-

DTPA and DMPE-DTPA-indium

3.8.1 Preparation of indium-111 radiolabeled indium citrate solution

5 μL of 111indium citrate solution (approximately 4 - 5 μCi) was dissolved in 1.25 mL

of saline. 0.2 mL of 111indium citrate saline solution was injected intravenously to each

mouse (a group of 5) through tail vein. The mice were euthanized by cervical

dislocation at time point 30 minutes post injection.

30

3.8.2 Preparation of indium-111 radiolabeled indium-DTPA solution

0.02 mg of DTPA was dissolved in 1.25 mL of saline. The solution was incubated with

5 μL of 111indium citrate solution (about 4 - 5 μCi) for 1 hour at RT, to allow for the

transchelation of indium-111. 0.2 mL of 111indium-DTPA saline solution was injected

intravenously to each mouse (a group of 5) through tail vein. The mice were euthanized

by cervical dislocation at time point 1 hour post injection.

3.8.3 Preparation of indium-111 radiolabeled DMPE-DTPA-indium solution

0.02 mg of DMPE-DTPA was dissolved in 1.25 mL of ethanol-emulphor-saline (1:1:18).

The solution was incubated with 5 μL of 111indium citrate solution (about 4 - 5 μCi) for

1 hour at RT, to allow for the transchelation of indium-111. 0.2 mL of DMPE-DTPA-

111indium solution was injected intravenously to each mouse (a group of 5) through tail

vein. The mice were euthanized by cervical dislocation at time points 30 minutes and 2

hours post injection.

3.8.4 In vivo Biodistribution Studies

For all euthanized mice, blood, urine and other solid organs (brain, heart, liver, lung,

spleen, kidney, testis, fat, bone, muscle and skin) were collected. Each sample was

carefully weighed and counted for indium-111 radioactivity as CPM using the auto-

gamma counter.

31

3.9 Biodistribution studies of iron-59 and carbon-14 oleic acid radiolabeled

PEGylated SPIONs

3.9.1 Preparation of iron-59 or carbon-14 oleic acid radiolabeled SPIONs

Particles radiolabeled with iron-59 in the SPION core were prepared by adding a small

volume (15 μL; 15 μCi) of radioactive 59Fe3+ chloride solution to the mixture of ferrous

and ferric chloride solutions before addition of ammonia (see procedure 3.3 for

preparation of unlabeled particles, earlier in this section). Similarly, particles

radiolabeled with carbon-14 oleic acid were prepared by adding a small volume (0.25

mL; 25 μCi) of carbon-14 oleic acid solution to the oleic acid that was added after

addition of ammonia. In both cases, neither the additional volume nor the extra mass of

material should have any effect on the formation and coating of the particles, since this

is being done in a volume of 45 mL using approximately 250 mg of iron and 100 mg of

oleic acid. The radioactivity in the preparation of iron-59 labeled SPIONs was estimated

using the ionization chamber. The radioactivity in a 10 μL sample of the carbon-14 oleic

acid labeled SPIONs was measured using a liquid scintillation counter (LS6500,

Beckman Coulter) since carbon-14 emits no photonic radiation.

After the formulation of iron-59 and carbon-14 oleic acid labeled SPIONs, the

PEGylated SPIONs were prepared as described above in section 3.4 with one difference.

The PEGylated particles were rehydrated using 1.5 mL saline instead of HPLC grade

water.

32

3.9.2 In vivo biodistribution studies

0.2 mL of iron-59 labeled PEGylated SPIONs saline solution or 0.2 mL of carbon-14

oleic acid labeled PEGylated SPIONs saline solution was injected intravenously to each

mouse (a group of 5) through tail vein. The mice were euthanized by cervical

dislocation at time points 10 minutes, 30 minutes and 24 hours post injection with five

mice in a group for each time points. Besides, a comparison group of five mice was

intravenously injected with 0.2 mL of ethanol-emulphor-saline (1:1:18) which only

contained about 0.3 μCi carbon-14 oleic acid.

For mice administered with iron-59 labeled particles, blood, urine and other solid

organs (brain, heart, liver, lung, spleen, kidney, testis, fat, bone, muscle and skin) were

collected. Each sample was carefully weighed and counted for iron-59 radioactivity as

count per minute (CPM) using the auto-gamma counter with appropriate energy

windows (800-1600 keV).

For mice administered with carbon-14 oleic acid labeled particles, blood, urine and

other solid organs (brain, heart, liver, lung, spleen, kidney, and testis) were collected.

Tissues were completely dissolved in SolvableTM and bleached with 30% hydrogen

peroxide before addition of UltimaGoldTM XR liquid scintillation fluid. The samples

were then assayed for carbon-14 using the liquid scintillation counter.

33

4. RESULTS AND DISCUSSIONS

4.1 Preparation and Characterization of PEGylated SPIONs

The PEGylated SPIONs were stable and did not show signs of aggregation after 1 week

at 4 °C (Figure 7). The size of the PEGylated SPIONs, as well as DTPA anchored

PEGylated SPIONs, was generally in the range of 30 - 50 nm (40.08 ± 6.75 nm, Figure

6). The surface zeta potential for the nanoparticles were always negative at about -36.6

± 6.58 mV. TEM images of the iron core of PEGylated SPIONs are shown in figure 8.

The size distribution of the iron core ranged from 5 nm to 25 nm. These results are in

agreement with previous studies (Sawant, 2008; Plouffe et al., 2011).

Figure 6. Particles size of PEGylated SPIONs measured using dynamic light scattering

in 90Plus particle size analyzer.

34

Figure 7. Stability Measurement of PEGylated SPIONs for consistent 7 days. Values are

mean ± s. d. (n = 5)

Figure 8. TEM micrograph of the iron core of PEGylated SPIONs.

05

101520253035404550

0 1 2 3 4 5 6 7 8

Mea

n N

ano

par

ticl

es S

ize

(nm

)

Time (Days)

Stability Measurement of PEGylated SPIONs

35

4.2 Radiolabeling of PEGylated SPIONs with Indium-111

The majority of the 111indium citrate used in the reaction mixtures was recovered in the

DTPA modified PEGylated SPIONs. Table 1 shows counting data from filters and

filtrates for successive centrifugations. The labeling efficiency percentage was higher

than 70%.

Table 1. Radioactivity recovered of 111indium with SPIONs in successive filtration. Values

are mean ± s. d. (n = 5)

111Indium Radioactivity (µCi)

Recovered from membrane Filtrate

First Centrifugation 7.7 ± 1.3 0.6 ± 0.4

Second Centrifugation 7.6 ± 1.1 0.1 ± 0.05

Third Centrifugation 7.6 ± 0.8 <0.1

4.3 Biodistribution of indium-111 radiolabeled PEGylated SPIONs

Biodistribution studies were conducted at 5 time-points after administration of indium-

111 labeled SPIONs: 10 minutes; 30 minutes; 1 hour; 2 hours; and 24 hours. The results

(percent injected radioactivity per gram tissue wet weight) are shown in Table 2. Liver

followed by spleen exhibited the highest radioactivity concentrations at all times.

Radioactivity in the blood decreased with time after administration, as to a lesser degree

did radioactivity in lung and heart, probably reflecting the large amounts of blood in

these organs. In contrast, radioactivity in liver increased with time. This rise was

significant (p<0.05) between 10 and 30 min. This is consistent with the notion that

indium-111 remained bound to nanoparticles in blood during the period of our

experiments, and that the particles continued to be accumulated by liver

36

reticuloendothelial cells, and remained trapped in these cells. Similarly to the case with

liver, the concentration of radioactivity in spleen and bone did not decrease over the

course of our experiments, again consistent with trapping by reticuloendothelial cells

in these organs (Martindale, Papadimitriou, & Turner, 1980) (Figure 9). Radioactivity

concentrations decreased in the order skin > muscle > testes > fat > brain, and also fell

between earlier and later time-points in these tissues. It is likely that all radioactivity

measured in the brain was actually in the brain vasculature, since it was very low at all

times, and its concentration was 3-5% of that in the blood at all time-points (Figure 11).

Kidney had the third highest concentrations of radio-indium at all times, after liver and

spleen; this could reflect trapping of smaller nanoparticles (HD < 10 nm) in the

glomerulus (Zuckerman & Davis, 2013). Some radio-indium was recovered in urine,

indicating that some loss of indium from the particles occurs (Figure 10).

Table 2. Biodistribution data of indium-111 labeled PEGylated SPIONs at different time

points (represented as the average percentage of injected activity per gram of each organ).

Values are mean ± s. d. (n = 5)

Tissues/ Times 10 minutes 30 minutes 1 hour 2 hours 24 hours

Liver 27.04 ± 1.89 33.90 ± 1.44 33.35 ± 1.12 34.83 ± 0.57 37.28 ± 1.03

Spleen 15.46 ± 0.41 15.71 ± 1.50 15.18 ± 1.06 13.43 ± 0.45 21.48 ± 2.41

Kidney 10.72 ± 1.57 7.71 ± 1.37 5.68 ± 0.91 9.57 ± 0.97 8.03 ± 1.00

Blood 9.59 ± 1.38 4.23 ± 0.81 4.22 ± 0.72 3.97 ± 0.58 0.55 ± 0.13

Lung 4.85 ± 1.01 2.99 ± 0.28 2.76 ± 0.50 2.65 ± 0.15 1.27 ± 0.31

Heart 2.32 ± 0.62 1.24 ± 0.16 1.08 ± 0.07 1.10 ± 0.18 0.62 ± 0.15

Bone 2.10 ± 0.19 3.14 ± 0.17 3.43 ± 0.40 3.66 ± 0.34 3.87 ± 0.25

Skin 1.36 ± 0.37 1.06 ± 0.07 0.73 ± 0.09 0.78 ± 0.20 1.00 ± 0.18

Muscle 0.80 ± 0.24 0.65 ± 0.10 0.41 ± 0.07 0.58 ± 0.09 0.53 ± 0.12

Testis 0.40 ± 0.05 0.32 ± 0.09 0.34 ± 0.05 0.42 ± 0.04 0.45 ± 0.07

Fat 0.30 ± 0.03 0.29 ± 0.05 0.16 ± 0.01 0.26 ± 0.01 0.22 ± 0.04

Brain 0.25 ± 0.09 0.13 ± 0.03 0.12 ± 0.02 0.11 ± 0.03 0.05 ± 0.01

Urine 11.17 ± 3.63 5.84 ± 0.97 2.47 ± 0.62 0.73 ± 0.34 7.83 ± 1.91

37

Figure 9. Graph of biodistribution of 111indium labeled PEGylated SPIONs at different

time points (represented as the average percentage of injected activity per gram of each

organ). Mice (25-30 g) were injected intravenously with indium-111 labeled particles

suspended in 0.2 mL of 0.9% NaCl solutions.

Figure 10. Indium-111 in urine after administration of 111indium labeled PEGylated

SPIONs.

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Biodistribution of 111indium labeled SPIONs at different time points

10 min

30 min

1 hour

2 hours

24 hours

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Biodistibution of 111Indium Labeled SPIONs in Urine

38

Figure 11. Graphs showing liver-to-blood (left) and brain-to-blood (right) ratios versus

time.

4.4 Biodistribution of indium-111 radiolabeled indium citrate, indium-DTPA and

DMPE-DTPA-indium

Surprisingly, the distribution of label from DMPE-DTPA-indium at 2 h was similar to

the distribution of label from 111indium labeled PEGylated SPIONs at that time, when

tissues were ordered according to concentrations of label. However, liver, spleen and

bone levels were lower for DMPE-DTPA-indium, while the blood, heart and lung

concentrations were higher. At the earlier time-point of 30 min, these differences from

the distribution seen for indium-SPIONs were more marked, and the concentrations in

liver and spleen were only half for DMPE-DTPA-indium than for indium-SPIONs,

while the blood level was 4 times higher for DMPE-DTPA-indium (Figure 12). At 30

min time point, the liver-to-blood ratio for DMPE-DTPA-Indium was <1, whereas for

indium-SPIONs it was > 7 (Figure 13).

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10 min 30 min 1 h 2 h 24 h

Live

r to

Blo

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Rat

io

Time

Liver to Blood Ratios versus Time

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0.15

0.20

10 min 30 min 1 h 2 h 24 h

Bra

in t

o B

loo

d R

atio

Time

Brain to Blood Ratios versus Time

39

Figure 12. Comparison of biodistribution between indium-SPIONs and DMPE-DTPA-

indium at 30 min and 2 h.

Figure 13. Comparison of liver-to-blood ratios versus time between indium-SPIONs and

DMPE-DTPA-indium.

Together, the data at 30 min and 2 h for DMPE-DTPA-indium indicate a much slower

transfer of label from blood to organs than for indium-SPIONs. It can therefore be

inferred that our preparations of indium-SPIONs do not contain appreciable amounts

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Comparison of Biodistribution between indium-SPIONs and DMPE-DTPA-indium at 30 min and 2 h

Indium-SPIONs 30 min

DMPE-DTPA-indium 30 min

Indium-SPIONs 2 h

DMPE-DTPA-indium 2 h

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Live

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Liver to Blood Ratio versus Time

Indium-SPIONs

DMPE-DTPA-indium

40

of free DMPE-DTPA-indium, and that the labeled phospholipid remains bound to the

SPIONs during the distribution phase. Nevertheless, DMPE-DTPA-indium could

dissociate from the SPIONs to some extent in the blood, and extensive dissociation

could occur in tissues after uptake, since the distribution of DMPE-DTPA-indium at 2

h is similar to that of indium-SPIONs; presumably, then, labeled phospholipid released

from indium-SPIONs would remain in the tissues where the nanoparticles were

deposited.

Two hours after administration of labeled substance, urine contained about 10 times

more radioindium for DMPE-DTPA-indium than for indium-SPIONs (Figure 14).

Thus, indium incorporated into the PEGylated SPIONs is more resistant to loss of a

radioactive species that can exit tissues and undergo clearance by the kidneys.

Figure 14. Comparison of urine radioactivity between indium-SPIONs and DMPE-

DTPA-indium.

Thirty minutes after administration of 111indium citrate, blood and kidney, as well as

bone, contained higher concentrations of radioactivity than after administration of

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Radioindium in Urine

Indium-SPIONs

DMPE-DTPA-indium

41

indium-SPIONs, while liver and spleen contained less (Figure 15). Indium is tightly

complexed by the iron-transport protein transferrin, and the observed disposition of

label may reflect a combination of the behaviors of Indium3+ and indium-transferrin.

Although this is speculative, one could suggest that the high blood level is due to

indium-transferrin, while the high concentration in bone is due to incorporation of

Indium3+ into the bone marrow, or to formation of indium oxide colloid that is taken up

by reticuloendothelial cells (Ebbe et al., 1996). Irrespective of the chemical forms

present, this experiment supports the notion that the tissue dispositions seen with

DMPE-DTPA-indium and indium-SPIONs are not predominantly due to Indium3+

released from its chelated form. This notion is strongly supported by examination of

the distribution of labeled form 111indium-DTPA at 1 h time point. Tissue concentrations

of label were quite distinct from those seen when indium citrate was injected. As

expected from the nuclear medicine literature on simple metal chelates, it was

characterized by extensive urinary excretion (Corcoran et al., 2010; Fritsch et al., 2009).

Tissue concentrations of radiolabeled 111indium-DTPA at 1 h were much lower than

obtained with the other forms of radio-indium. Kidney had the highest level, probably

reflecting indium chelate in the urinary collection system (Corcoran et al., 2010; Fritsch

et al., 2009).

42

Figure 15. Comparison of biodistribution of indium-SPIONs, DMPE-DTPA-indium and

indium citrate at 30 minutes post injection, showing distinct distribution patterns for each

form of radioactive indium. .

Table 3. Biodistribution data of indium-111 labeled DMPE-DTPA-indium, indium citrate

and indium-DTPA (represented as the average percentage of injected activity per gram of

each organ). Values are mean ± s. d. (n = 5)

Tissues DMPE-DTPA-

111Indium (30 min)

DMPE-DTPA-111Indium (2 h)

111Indium citrate

(30 min)

111Indium-

DTPA (1 h)

Liver 16.10 ± 1.19 25.27 ± 0.42 20.58 ± 2.79 0.23 ± 0.06

Spleen 8.11 ± 1.21 7.03 ± 0.37 8.19 ± 1.60 0.13 ± 0.02

Kidney 4.79 ± 0.49 5.14 ± 0.60 27.33 ± 3.46 2.16 ± 0.65

Blood 18.60 ± 0.92 8.07 ± 0.91 14.58 ± 3.26 0.39 ± 0.17

Lung 11.19 ± 1.19 7.53 ± 0.77 10.73 ± 1.98 0.41 ± 0.06

Heart 4.57 ± 0.38 2.65 ± 0.33 4.22 ± 1.39 0.23 ± 0.13

Bone 1.79 ± 0.29 1.90 ± 0.37 6.72 ± 1.02 0.82 ± 0.35

Skin 0.55 ± 0.19 0.77 ± 0.12 2.35 ± 0.65 0.48 ± 0.09

Muscle 0.61 ± 0.04 0.54 ± 0.07 2.22 ± 0.83 0.27 ± 0.16

Testis 0.50 ± 0.07 0.59 ± 0.06 1.05 ± 0.21 0.35 ± 0.33

Fat 0.16 ± 0.06 0.28 ± 0.08 1.20 ± 0.31 0.13 ± 0.07

Brain 0.53 ± 0.06 0.30 ± 0.17 0.51 ± 0.12 0.05 ± 0.02

Urine 7.74 ± 2.16 9.45 ± 2.37 16.53 ± 3.46 79.30 ± 37.75

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Biodistribution of Indium-SPIONs, DMPE-DTPA-indium and Indium citrate at 30 minutes Time Point

Indium-SPIONs

DMPE-DTPA-indium

Indium citrate

43

4.5 Biodistribution of iron-59 or carbon-14 oleic acid radiolabeled PEGylated

SPIONs

The incorporation efficiency for iron-59 was approximately 53% (8 μCi out of 15 μCi),

assayed using the ionization chamber, while the carbon-14 oleic acid incorporation

efficiency was about 8% (2 μCi out of 25 μCi), estimated by the liquid scintillation

counter. The poor efficiency for labeling with carbon-14 is due to the large excess of

oleic acid over iron salts used in the preparation of the SPIONs.

When the iron core of the SPIONs was labeled with iron-59, the distribution pattern at

each time-point was marked by higher fractions of injected radioactivity in liver and

spleen than for indium-111 (compare Table 2 with Table 4), and lower concentrations

elsewhere. However, bone, kidney, blood and urine contained more radioactivity than

other organs (except liver and spleen).

The fraction of injected iron-59 in liver decreased by about 15%, from 55% to 45%

between 10 min and 24h (p < 0.05). This decrease, together with the finding of iron-59

in urine (as well as increased levels of radioactivity in kidney and bone) suggests that

PEGylated SPIONs in the liver can be slowly broken down to one or more soluble iron

species.

44

Table 4. Biodistribution data of iron-59 labeled PEGylated SPIONs (represented as the

average percentage of injected activity per gram of each organ). Values are mean ± s. d.

(n = 5)

Tissues/Time 10 minutes 30 minutes 24 hours

Liver 54.58 ± 0.87 51.16 ± 2.95 45.04 ± 2.10

Spleen 17.83 ± 0.65 17.78 ± 0.63 17.36 ± 1.45

Kidney 0.40 ± 0.18 0.43 ± 0.14 0.72 ± 0.21

Blood 1.32 ± 0.19 0.44 ± 0.09 1.26 ± 0.43

Lung 0.52 ± 0.03 0.61 ± 0.07 0.19 ± 0.03

Heart 0.14 ± 0.09 0.29 ± 0.11 0.19 ± 0.09

Bone 0.95 ± 0.24 1.34 ± 0.47 1.79 ± 0.53

Skin 0.17 ± 0.10 0.04 ± 0.45 0.21 ± 0.07

Muscle 0.04 ± 0.03 0.16 ± 0.31 0.35 ± 0.14

Testis 0.00 ± 0.02 0.30 ± 0.08 0.07 ± 0.07

Fat 0.02 ± 0.01 0.04 ± 0.08 0.10 ± 0.05

Brain 0.02 ± 0.01 0.04 ± 0.03 0.04 ± 0.01

Urine 0.27 ± 0.17 0.76 ± 0.29 0.73 ± 0.22

Together, the data for indium and iron labeled particles suggest that during the

distribution phase about half of the radioactivity dissociates from the particles in the

form of DMPE-DTPA-Indium. However, as demonstrated in the experiment where

DMPE-DTPA-indium itself was administered, much of this radioactivity can be

subsequently taken up and retained by the liver and spleen. Thus, at 10 minutes post

injection the radioiron/radioindium ratio in liver is 2:1, but due to recirculation of

DMPE-DTPA-indium at 24h it has decreased to 1.25 (Figure 16).

When particles labeled with carbon-14 oleic acid were injected into mice, a different

pattern of distribution (Table 5) was observed than when the particles were labeled with

iron-59. Although the liver contained the highest concentration of carbon-14 at 10 and

30 minutes post injection, this halved (18 % IA/g to 9 % IA/g) between the two early

45

time-points. Spleen, blood, kidney, heart and lung all contained approximately equal

concentrations of carbon-14 (about 3% IA/g) at both 10 and 30 minutes, while the

concentrations in testis and brain were much lower (about 1% IA/g). Carbon-14 levels

in urine were higher than those in non-hepatic tissues at 10 and 30 minutes. By 24 hours

after administration of labeled particles, radioactivity-concentrations in all tissues,

including liver, were low (< 2% IA/g).

Table 5. Biodistribution data of carbon-14 oleic acid labeled PEGylated SPIONs

(represented as the average percentage of injected activity per gram of each organ). Values

are mean ± s. d. (n = 5)

Carbon-14 oleic acid labeled PEGylated SPIONs Carbon-14 oleic

acid only

Tissues/Time 10 minutes 30 minutes 24 hours 30 minutes

Liver 17.54 ± 1.71 9.47 ± 0.86 1.24 ± 0.16 1.80 ± 0.64

Spleen 3.16 ± 0.40 2.53 ± 0.20 1.27 ± 0.24 0.50 ± 0.19

Kidney 4.24 ± 0.65 3.17 ± 0.27 1.27 ± 0.13 0.85 ± 0.15

Blood 3.07 ± 0.75 3.47 ± 1.17 1.53 ± 0.21 0.60 ± 0.20

Lung 3.55 ± 0.56 3.29 ± 1.10 0.89 ± 0.38 3.52 ± 0.82

Heart 3.55 ± 1.31 2.73 ± 0.44 0.45 ± 0.13 1.50 ± 0.37

Testis 0.68 ± 0.08 0.64 ± 0.08 0.54 ± 0.15 0.16 ± 0.07

Brain 0.50 ± 0.05 0.40 ± 0.07 0.30 ± 0.03 0.11 ± 0.03

Urine 11.47 ± 1.05 10.03 ± 1.22 0.88 ± 0.24 0.80 ± 0.11

These observation, compared to those made using iron-59 labeled particles,

demonstrate that oleic acid does not remain attached to the iron oxide core of the

particles, in vivo. The failure to see high uptake in the spleen even at 10 minutes

indicates that dissociation is quite rapid. The higher uptake in liver than other organs at

10 and 30 minutes may reflect initial storage in complex lipid pools in this organ, which

is followed by incorporation into lipoproteins for export to other tissues via the blood.

46

Other processes such as partial oxidation to ketone bodies (acetoacetate and beta-

hydroxybutyrate), again exported to other tissues, are also expected to occur.

Figure 16. Comparison of indium-111, iron-59, and carbon-14 oleic acid biodistribution

patterns in liver, spleen, bone, blood and kidney at 10 min, 30 min, and 24 h time points.

(a)

(b)

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111Indium-SPIONs

59Iron-SPIONs

14Carbon-SPIONs

0.00

5.00

10.00

15.00

20.00

25.00

30.00

10 min 30 min 24 h

Ave

rage

%In

ject

ed A

ctiv

ity/

g

Time

Spleen

111Indium-SPIONs

59Iron-SPIONs

14Carbon-SPIONs

47

(c)

(d)

(e)

0.00

0.50

1.00

1.50

2.00

2.50

3.00

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4.50

10 min 30 min 24 h

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rage

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ject

ed A

ctiv

ity/

g

Time

Bone

111Indium-SPIONs

59Iron-SPIONs

0.00

2.00

4.00

6.00

8.00

10.00

12.00

10 min 30 min 24 h

Ave

rage

%In

ject

ed A

ctiv

ity/

g

Time

Blood

111Indium-SPIONs

59Iron-SPIONs

14Carbon-SPIONs

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

10 min 30 min 24 h

Ave

rage

%In

ject

ed A

ctiv

ity/

g

Time

Kidney

111Indium-SPIONs

59Iron-SPIONs

14Carbon-SPIONs

48

Biodistribution data for SPIONs labeled with iron-59, indium-111 and carbon-14 which

are discussed above for each radionuclide are re-displayed in Figure 16 for tissues, (a)

liver, (b) spleen, (c) bone, (d) blood and (e) kidney, at 10 min, 30 min and 24h. This

facilitates comparison of the three radionuclides in terms of their effectiveness in

labeling SPIONs. (Panel (c) for bone shows data for iron-59 and indium-111 only since

carbon-14 levels were too low to be measured in bone.) These comparisons illustrate

the points made above that while indium-111 is not a perfect proxy for iron-59, it might

be useful for imaging the distribution of SPIONs using SPECT. However, it is clear that

the oleic acid component of the SPIONs does not remain with the iron oxide core after

administration to mice.

As to the biodistribution of carbon-14 oleic acid alone, it is very different from the 14C-

SPIONs at 30 minutes time point. Less radioactivity remained in tissues (except lung)

after administration of oleic acid alone than after administration of carbon-14 oleic acid

labeled SPIONs (Table 5). One explanation is that, in associating with and dissociating

from the SPIONs, most or perhaps all of the carbon-14 oleic acid is converted to a

chemical form that is cleared more slowly from the body. Oleic acid itself may be

cleared rapidly because long chain fatty acid are a major metabolic fuel and are rapidly

converted to labeled carbon dioxide, which leaves the body in exhaled breath.

Additionally, there is more radioactivity in lungs than other tissues after injection of

14C-oleic acid alone, whereas there is more radioactivity in liver than other tissues after

administration of carbon-14 oleic acid labeled SPIONs. Some compounds that are very

49

lipophilic are trapped in the lungs immediately after intravenous administration because

they dissolve in the lung surfactant and oleic acid is one of these compounds (Koren et

al., 1980). If this is the explanation for the higher uptake in lungs, we could argue that

the failure to see higher lung uptake from oleic acid labeled SPIONs is consistent with

the view that the carbon-14 released from the SPIONs is in a different chemical form.

Thus, radiochromatographic studies on tissues will be necessary to confirm these

speculations.

4.6 OVERALL CONCLUSIONS

After intravenous injection of radiolabeled particles, the radioactive indium was

preferentially localized in those organs (liver, spleen and bone) associated with trapping

of colloids by reticuloendothelial cells, with no reduction of radioactivity concentration

up to the longest time-point examined (24h).

The tissue distributions obtained with indium-111 SPIONss at various times are

encouraging for use of such labeled particles in imaging experiments. The differences

in distribution of either indium-citrate or of indium-DTPA from those obtained with

labeled SPIONs supports the view that a substantial fraction of the indium-111 remains

associated with the iron oxide core. However, the somewhat similar distribution of

DMPE-DTPA-indium to that of indium-111 labeled SPIONs, in terms of high uptake in

organs containing reticuloendothelial cells, raises the question of whether some of the

uptake seen with labeled SPIONs in liver and spleen is due to recirculation of

dissociated DMPE-DTPA-indium to these organs. In other words, if this labeled

50

phospholipid itself preferentially distributes to liver and spleen, then the fact that

indium-111 from the labeled SPIONs is found in liver and spleen does not absolutely

prove that the labeled particles remain intact in vivo.

In contrast to the situation with indium-111, it is clear that carbon-14 oleic acid

incorporated into the SPIONs does not remain firmly associated with the iron oxide

core. Carbon-14 appears to be released rapidly from the SPIONs in the blood since the

spleen is not targeted by carbon-14 labeled SPIONs, though this organ is targeted by

SPIONs labeled with indium-111 or iron-59. However, the carbon-14 released does not

appear to be in the chemical form of oleic acid, since the biodistributions of radiolabel

administered as oleic acid itself or oleic acid labeled SPIONs were quite distinct.

51

5. LIMITATIONS AND FUTURE DIRECTIONS

PEGylated SPIONs that were characterized by DLS and TEM were not radioactive, as

we were not allowed to use radioactive materials in these analytical instruments.

The procedures used to prepare SPIONs radiolabeled with iron-59 and with carbon-14

oleic acid, addition of labeled reagent to the reaction mixture conducted on the usual

scale, limited the radioactivity of carbon-14 or iron-59 that could be injected into a

mouse. We were able to deal with this problem by using long counting times. However,

if further studies with these radionuclides are warranted, one could improve

radiochemical yields and specific activities by miniaturizing the mass scale of the

syntheses but maintaining the same amounts of radioactive iron or oleic acid. However,

it is very likely that the size and possibly other characteristics of the SPIONs may

depend on factors such as heating rates that could be altered by the scale of the reaction,

so that miniaturization might require extensive development work.

Further progress in understanding the behavior of the indium-111 labeled PEGylated

SPIONs will require analysis of the chemical form(s) of indium present in tissues

(including liver, spleen and especially kidney and urine) using radiochromatographic

methods.

52

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