Chapter 18: MRI in Nanotechnology: Nanomagnetic Probes for Bioimaging

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006).

Chapter 18: MRI in Nanotechnology: Nanomagnetic Probes for

Bioimaging

R.V. Ramanujan*, C.K. Tan and H. Rumpel

Singapore General Hospital, Singapore 169608 *School of Materials Science and Engineering

Nanyang Technological Univerity, Singapore 639798

INTRODUCTION

This article describes some recent work being performed to evaluate nanomagnetic

particles for combined bioimaging and drug targeting applications in the context of

human cancer treatment. The synthesis, characterization and property evaluation of

such particles is described and representative MRI results are provided.

Magnetic materials in particle form have been used for a variety of biomedical

applications.[1-5] One such applications is in the area of drug delivery, where coated

magnetic nanoparticles (called carriers) have been used. Such carriers can show

strong contrast-enhancing abilities in Magnetic Resonance Imaging (MRI), they

typically consist of a magnetic particle core, e.g., of iron, coated with a polymer in

which drugs are dissolved. Pharmacological trials on human subjects using this

approach have been undertaken and the methodology has received the Fast Track

Designation of the US Federal Drug Administration. We are developing magnetic

carriers for anti-cancer drug targeting in human liver cancer treatment. Key

parameters for drug delivery by this approach are the efficiency of targeting, the

carrier concentration and the distribution of the particles in the body.

Three factors must be considered in the design of the particles: to reduce toxicity, iron

concentration must be minimized, which will, however, adversely affect the visibility

by MRI. To increase the drug load of the particles, coating thickness must be

increased, since the drug is dissolved in the coating. However, an increase in coating

thickness will decrease the targeting efficiency. Hence we need to study the influence

of these factors on the particle’s efficiency for drug targeting and imaging.

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006). In this study, the drug delivery efficacy of our synthesized particles was assessed by

determining the biodistribution of intra-arterial administered particles in rat

hepatocellular carcinoma (HCC) using MRI. Morris hepatoma cell line is implanted

via laparotomy into the liver of buffalo rats to induce development of HCC. A

solution of magnetic nanoparticles is then delivered via the hepatic artery and MRI

performed to check the localization of the carriers in the HCC. It was found that the

particle parameters conventionally employed for drug targeting (concentration, size

and type of magnetic particle and especially the coating thickness and composition)

differs from the parameters needed for optimal MR-imaging. Hence the need to

concurrently examine those parameters for the design of iron-based coated magnetic

particles required for optimized drug targeting and MR imaging. The novelty is the

integration of magnetic drug targeting with‘in vivo’ bioimaging in order to assess the

efficacy of drug delivery and the biodistribution of carriers.

The methodology to be followed is :

• synthesis and characterization of nanomagnetic carriers

• ‘in vivo’ observations using MR imaging and histopathology

• Assessment of drug targeting efficiency and correlation to localization

Chemotherapy and radiotherapy are conventional methods of cancer treatment.

These treatment modes typically have undesirable side effects, and as a result, limit

the dose of anti cancer drugs that can be delivered to the tumor site. One way to

circumvent this problem is to use materials that will take the drug only to the tumor

and then release the drug, ideally in a controlled fashion, or more realistically, in a

triggered fashion. This can be achieved by making the magnetic particles and coating

them with a polymer coat, in which anti cancer drugs are dissolved. Such a materials

system is called a carrier. When these carriers are injected in the blood stream and a

large magnetic field gradient is generated at the tumor locus, it is found that the

carriers are attracted by the magnetic field and are retained at the tumor. At the tumor

site, the carriers can release anti cancer drugs. Ideally, we would like to have a small

magnetic core and a large coating thickness since the anti cancer drug is dissolved in

the coating. However, the smaller the magnetic core, the lesser will be the targeting

efficiency. Hence the key issues are: How efficient is this targeting? What happens to

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006). the carriers after targeting and drug release, i.e., what is the biodistribution? Can we

find out where these particles are going by bioimaging techniques?

It is proposed to develop bioimaging of magnetic carriers that can be used for drug

delivery applications for cancer treatment. MRI investigations of these carriers are

undertaken to determine the concentration and biodistribution of such carriers in the

context of liver cancer treatment studies (human hepatocellular carcinoma in a rat

model). It would be of interest to find out as to what would be the effect of coating

thickness and composition on the MR imaging and where the carriers are distributed

as a function of time.

From a toxicity point of view, it is desirable to minimize the concentration of iron

used and increase the coating thickness, since the drug is dissolved in the coating.

However, an increase in coating thickness will decrease the targeting efficiency.

Hence there is a need to study the effect of coating thickness and composition on the

targeting efficiency and MRI based detection of coated nanomagnetic carriers. In

addition, it is found that the concentration, size and type of material and especially the

coating thickness and composition conventionally used for drug targeting differs from

that needed for optimum imaging. It is, therefore, necessary to concurrently examine

the requirements of drug targeting and MRI imaging to establish the optimum

concentration to be used for such studies.

The specific aims are:

(a) To study the effect of coating thickness and composition on MRI based detection

of coated nanomagnetic carriers used in targeted magnetic drug delivery for cancer

treatment. Such studies directly relate to the efficiency of drug targeting.

(b) To determine the concentration and biodistribution of such carriers in ongoing

investigations being carried out in the context of ‘in vivo’ imaging by MRI of the

localization of such carriers.

The novelty is the integration of magnetic drug targeting with ‘in vivo’ bioimaging

experiments in order to assess the efficacy of drug delivery and the biodistribution of

the carriers.

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006). The work is significant because an integrated approach to the development and

testing of magnetic carriers which can be used for drug delivery studies is being

undertaken.

The likely applications of this work are obvious and important. It is clear that the

development of MRI techniques to study magnetic nanoparticle uptake into diseased

regions, such as tumors, will be very useful in a wide variety of pre-clinical studies as

well as in clinical trials.

From a technological viewpoint the development of MRI related instrumentation and

software, specific to ‘in vivo’ studies of drug delivery is a much needed tool for

studies in an animal model. The development of magnetic carriers that can be useful

for drug delivery as well as for bioimaging will be useful not only for disease

treatment but also for fundamental developmental studies. From a scientific point of

view a fundamental understanding of the role of the coating thickness and

composition on the image contrast will help us to develop a wider range of MRI

contrast agents.

The societal applications of this work are that it provides a diagnostic tool for a

variety of disease treatment and biomarker applications. The present focus is on

cancer treatment, however, aspects related to surveillance and detection of other

diseases are equally important.

The economic impact goes beyond high quality magnetic nanocarriers for drug

delivery applications. Materials and methods can be readily developed for

commercial applications, e.g., monitoring the tumor size during therapy, for reducing

the number of pre-clinical trials by ‘in vivo’ monitoring of the biodistribution of the

drugs as a function of time, as well as for improved MRI detection of cancer by other

suitable carriers. Preliminary work in this direction in case of breast cancer has been

performed by other groups.

METHODS

The overall methodology to be followed can be summarized as

• Preparation of carriers with systematic variation of carrier concentration and

polymer coating thickness


• MR Imaging of the carriers during ‘in vivo’ trials of drug targeting

• Assessment of localization and biodistribution of the carriers as a function of

particle type and coating thickness

This will lead to a fundamental understanding of the drug targeting efficiency using

MR imaging. It will also result in a determination of the optimum carrier

concentration, size and coating thickness for concurrent drug delivery and bioimaging

studies.

EXPERIMENTAL STUDIES

Synthesis Of Nanomagnetic Probe:

The preliminary studies consisted of the synthesis of a variety of nanomagnetic

particles (Figs. 1, 2) and some of these results have been reported earlier [6-11]. These

particles have been prepared by physical methods such as milling as well as chemical

synthesis methods such as reverse micelle and the polyol method. Polymer coatings

and drugs have been coated around these particles mainly by solvent evaporation

techniques.

Three studies conducted by us on such materials and based on Refs. 6 to 11 will be

discussed below:

Polymer Coated Ceramic Iron Oxide Particles: First, the iron oxide powder was

ball milled. Microspheres were then synthesized by solvent evaporation, resulting in

iron oxide particles encapsulated in a polymer and drug coating. Various parameters,

such as the duration of milling and agitation speed as well as the polymer

concentration were varied. A milling time of 72 h. was found to yield a small size and

narrow size distribution of particles; the average particle size was about 600 nm.

Measurements of the change in grain size and the magnetic properties of the powder

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006). with milling time were performed. The agitation speed and polymer concentration of

400 rpm and 0.04 g poly(l-lactic acid) in 8 g dicholoromethane respectively, was

found to yield small, spherical microspheres with a narrow size distribution. The

surface morphology and magnetic properties of the microspheres was also analyzed.

Polymer Coated Metallic Magnetic Particles: Cobalt and iron powders were ball

milled and subsequently characterized by SEM and XRD methods. The average size

of the ball milled Co powders was 7.4μm after 1 h. of milling, for Fe, the smallest

average size was 808 nm after 10 h. of milling. These milled powders were then

coated, using the solvent evaporation technique with the polymer poly-l-lactic acid to

form microspheres. The process parameters to form microspheres with the smallest

diameter, spherical morphology and a narrow size distribution was determined. The

stirring speed and polymer viscosity were found to control the size and shape of

microspheres. The Co and Fe microspheres had an average size of 105μm and 125μm,

respectively. The VSM results showed that saturation magnetization decreased with

polymer coating thickness.

Stimuli Sensitive Polymer Coated Magnetic Particles: Temperature-sensitive Poly

(N-isopropylacrylamide), PNIPA gels were synthesized with micron-sized iron and

iron oxide (Fe3O4) particles to investigate their viability for hyperthermia applications.

Induction heating of the magnetic hydrogels with varying concentration of magnetic

powder was conducted at a frequency of 375 kHz for magnetic field strength varying

from 1.7 kA/m to 2.5 kA/m. It was observed that the maximum temperature induced

in the magnetic hydrogels increased with the concentration of magnetic particles and

magnetic field strength. The PNIPA gel underwent a collapse transition at 340C. It

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006). was found that a 2.5 wt% Fe3O4 in PNIPA composite took 260s to be heated to 45oC

under a magnetic field strength of 1.7 kA/m, the specific absorption rate (SAR) was

found to be 1.83. SAR of iron oxide was found to be higher than the SAR of iron.

Fig. 1 Superparamagnetic particles


Fig. 2 polymer coated magnetic carriers [ref. 11]

PRELIMINARY MR STUDIES

MRI using contrast agents has been used for a variety of purposes in the area of

bioimaging [12-16], in this application a dedicated solenoid radiofrequency coil for

Magnetic Resonance (MR) imaging of rat liver was used in conjunction with a

whole-body MR scanner (see Figure 3 ). Fig 4 shows a typical result of MR studies

before and after administration of the magnetic particles. It can be seen that the solid

part of the tumor appears hypointense (T1 shortening) while the cystic part in the

centre of the tumor and the cavity (due to spillage of Fe-particles during

administration through the hepatic artery) show ‘signal void.’


Figure 3: Purpose-built coil used for preliminary MR studies


Figure 4: Before (left) and after administration of Fe-particles (right). Solid part

of the tumor appears hypointense (T1 shortening) while the cystic part in the

centre of the tumor and the cavity (due to spillage of Fe-particles during

Looking into Living Things through MRI, R.S. Chaughule and S.S. Ranade (eds.), pages 298-305, Prism Publications (2006). administration through the hepatic artery) show ‘signal void.’ (H. Rumpel, SGH,

Singapore)

CONCLUSIONS

It has been shown that nanomagnetic probes can, in principle, be used for combined

bioimaging and drug targeting and a number of advantages of this procedure have

been outlined. Nanoparticles can be synthesized and functionalized to target cancer

cells for use in the molecular imaging of a malignant lesion. Large numbers of

nanoparticles are injected into the body and they preferentially bind to the cancer cell,

defining the anatomical contour of the lesion and making it visible. These

nanoparticles give us the ability to see cells and molecules that we otherwise cannot

detect through conventional imaging. Combidex (ferumoxtran-10) is an

investigational functional molecular imaging agent, consisting of iron oxide

nanoparticles, that is currently being developed for use in conjugation with MRI

(Magnetic Resonance Imaging) to aid in the differentiation of cancerous nodes from

normal lymph nodes. It is administered via a 30-min infusion and it accumulates

preferentially in normal lymph node tissue, thus facilitating the differentiation

between malignant and nonmalignant lymph nodes. Further work is required to

optimize these parameters and create the best nanomagnetic probes.

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Chapter 18: MRI in Nanotechnology: Nanomagnetic Probes for Bioimaging

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