1. “Intracellular Magnetically modified Red Blood Cells as Contrast Agents in Magnetic
Resonance Imaging”
Abstract
Magnetic nanostructures have gained much attention in recent years owing to their
unique properties which make them attractive for utilization in several diagnostic
techniques. One of these is Magnetic Resonance Imaging (MRI) that employs magnetic
nanostructures, in specific nanoparticles of iron oxides (NIO), as contrast agents (CA), to
improve the contrast of the recorded images and therefore to improve the overall imaging
quality, that ultimately enables Radiologists to distinguish pathological from healthy tissues.
In comparison to other CA (e.g gadolinium), NIO have numerous advantages: they are more
biocompatible, have higher magnetic susceptibility though of equivalently small remanent
magnetization, thus avoiding the formation of cluster in the cardiovasacular system and
subsequent thrombosis complications. Besides those important advantages, NIO have a
major disadvantage in regards to their biodistribution and relevant kinetics, since they are
subjected to phagocytosis from the reticuloendothelial system and relatively quick excretion
from the urinary system. As a result NIO have a short half-life of circulation in the
cardiovascular system (few hours to few days).
In the present Master Thesis we aimed to counterbalance that limitation by
introducing a magnetically-modified cell line as possible CA . In specific, we focused on
erythrocytes (EC), for numerous reasons. Most importantly, EC have the longest lifespan
(120 days) in comparison to other cells of peripheral blood, that is leucocytes and plateletes.
There is also vast knowledge on EC in respect to their properties, since they have been
studied to a great extent. Finally, they can be easily collected from any donor in relatively
large amounts and can be easily processed in the lab. Specifically, as we show in this Master
Thesis EC can be subjected to magnetic modifications, thus we term them magnetically-
modified EC (mmEC).
We proposed two methods to produce mmEC: (A) By endocytosis of NIO: The NIO
that we used are magnetite/maghemite ( Fe3O4/Fe2O3) which are superparamagnetic
particles with properties that have been extensively studied in the literature. By replacing
part of EC’ cytoplasm with NIO we created a hybrid cell-magnetic carrier which has unique
characteristics. On the one hand, the embedded NIO empower the carrier EC with high
magnetization making them ideal CA in MRI applications. On the other hand, EC act as a
biocompatible host carrier for the NIO, thus protecting them from both being recognized by
the reticuloendothelial system and from being excreted from the urinary system. (B) By
reducing the intracellular hemoglobin of EC. It’s safe to assume that by reducing the EC’
hemoglobin we alter their magnetization, thus making them prospective CA for MRI
applications.
We note that in both cases (A) and (B), we used venous blood thus it was rich in
carbaminohemoglobin, which is a product of hemoglobin and has a paramagnetic behavior.

2. In both cases, (A) and (B), the modification of the magnetism of the EC’ cytoplasm
will alter the signal recorded in MRI, enhancing thus the contrast, in comparison to
surrounding tissues, of the recorded image. It is also safe to assume that the mmEC are
totally biocompatible and will not be removed by the reticuloendothelial and urinary system.
That would result a pronounced circulation half-life of the mmEC, in the cardiovascular
system, on the order of tens of days. Such long half-life would enable us to perform
successive imaging sessions of the cardiovascular system for long periods of time by using
the same CA.
The experimental procedure for the development of mmEC had three basic phases:
Phase (I): For both methods of magnetic modification, (A) and (B), it is essential to
reduce as much as possible the hemoglobin of the EC. This can be achieved with partial lysis-
by using aqueous solution of NaCl of suitably low tonicity x% (x=0.0-0.5%). Before that there
should be a stabilization of the EC’ cytoskeleton through maturation with physiological
saline, NaCl 0.9% [D.Stamopoulos unpublished results]. To find the optimum tonicity x% we
conducted systematic series of experiments by varying the tonicity of the NaCl x% (x=0.0-
0.5%). We thus defined that with x=0.2-0.3% we can achieve the partial (but not total) lysis
of the EC by creating pores in their membranes. The EC that are developed at the end of this
process are called partially lysed EC (plECs). The characteristic of their pores (population and
diameter) that are created on the membrane of the produced plEC were systematically
studied by proper microscopy techniques (see below).
Phase (II):After phase (I) and exclusively in regards to the first method of magnetic
modification, follows the endocytosis of NIO into the plECs through maturation under
adequate conditions. We note that the NIO must have proper characteristics to enter the
plEC. Obviously, their diameter should be smaller than the diameter of the pores that we
have created on the EC’ membrane. For that reason, we initially developed NIO from iron
salts (FeCl2 and FeCl3) through a conventional wet-chemistry procedure based on a rapid
reaction of co-precipitation that takes place at appropriate conditions of pH=11-12. To
remove cluster of NIO of high diameter that existed at the end of the reaction, we
developed homemade cotton-based filter columns by using standard suringes. The
characteristics of NIO (concentration and diameter) and their successful endocytosis in the
plEC, were studied, in detail, through proper microscopy techniques (see below).
Phase (III): In the final phase, plECs of both methods, (A) and (B), are resealed
through maturation under appropriate conditions in two candidate media. In specific, two
protocols were studied, the first wwas based on a natural medium (autologous plasma of
the donor) and the second was based on an artificial (human albumin in physiological saline
50g/L-Baxter®). Great emphasis was given to find the adequate duration of the maturation
to reseal the plEC successfully. The performance of each protocol was studied in detail
through proper microscopy techniques (see below).
In the present dissertation we used the following processing, characterization and
microscopy experimental techniques: (1) Centrifugation for the isolation of EC. (2)
Cytospinner for the development of single-layer films of blood cells onto standard
microscopy glass slides. (3) Optical microscope for the introductory evaluation of the

3. produced sindle-layer films of blood cells. (4) Atomic Force Microscope (AFM) and Scanning
Electron Microscope (SEM) that we used throughout all phases of the present investigations
due to their excellent spatial resolution on the order of nanometers (1 nm=10-9
m). (5)
Energy dispersive X-ray analysis (EDAX) for the elemental analysis of NIO and the evaluation
of their endocytosis in the plEC (6) Superparamagnetic Quantum Interference Device
(SQUID) magnetometry for the evaluation of the produced plEC.
In conclusion, in the experiments performed in the frame of the present
dissertation we produced mmEC with two methods (A) through partial replacement of
hemoglobin by NIO and (B) through reduction of the intracellular hemoglobin. The
evaluation of all phases with the basic techniques of AFM, SEM, EDAX and SQUID showed
that the laboratory protocols were standardized to a great extent. We suggest that the
mmEC can be used as a completely biocompatible CA in MRI, allowing the imaging of the
cardiovascular system for long time spans and improving significantly the contrast of the
recorded image. Besides diagnostic applications, mmECs can be used in therapeutic
applications, such as carriers of radio/chemical-therapeutic factors for their selective uptake
by pathogenic regions of interest, through magnetic guidance. Further research must be
conducted to optimize the whole process of mmEC development and also to study their
performance in both in vitro and in vivo applications, starting from the more simple
diagnostic procedures and continuing with the more complex therapeutic processes.