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Development of a method for bioanalysis of a nanomaterial in biological matrices

Fernández, Gema (2015) MOBN01 20151
Degree Projects in Molecular Biology
Abstract
Molecular imaging techniques have become an essential tool for cancer diagnosis. They have the potential to detect cancer at early stages and thus, they can change the outcome of the disease and patient prognosis. Magnetic resonance imaging (MRI) has arisen as one of the most promising imaging methods used for the screening of soft tumor tissues such as breast cancer. However, the specificity of MRI remains poor leading to misdiagnosis and many false positive findings. Thus, a wide range of new contrast agents (CAs) are being developed in order to enhance image resolution and safety in cancer diagnosis. For new drugs with an intended clinical use, it is necessary a deep insight into the viability of the molecule prior to clinical trials.... (More)
Molecular imaging techniques have become an essential tool for cancer diagnosis. They have the potential to detect cancer at early stages and thus, they can change the outcome of the disease and patient prognosis. Magnetic resonance imaging (MRI) has arisen as one of the most promising imaging methods used for the screening of soft tumor tissues such as breast cancer. However, the specificity of MRI remains poor leading to misdiagnosis and many false positive findings. Thus, a wide range of new contrast agents (CAs) are being developed in order to enhance image resolution and safety in cancer diagnosis. For new drugs with an intended clinical use, it is necessary a deep insight into the viability of the molecule prior to clinical trials. Some in vitro analysis and in vivo animal studies are carried out in order to characterize the physicochemical properties of the compound and its toxicity. Problems to find the right methodology to detect and assess identity of nanoparticles after being injected into the blood have been previously found. In this project, a combined method to extract and characterize dummy particles, mimicking nanoparticles to be used as CAs, was developed. These particles consist of a polymeric core, which contains a metal ion inside, together with a coating attached to the surface. A new approach for characterization was investigated by using three analytical techniques: gel permeation chromatography (GPC) to extract and separate the nanoparticles by size, enzyme-linked immunosorbent assay (ELISA) to analyze the coating and inductively coupled plasma optical emission spectroscopy (ICP-OES) to analyze the core and metal ion. Different biological matrices, mainly serum and urine, were tested to prove identity. Results showed an efficient extraction of the material with a high rate of recovery by GPC. Moreover, the accuracy and sensitivity of ELISA and ICP-OES in detecting the coating and composition of nanoparticles respectively was proved. More importantly, identity of the extracted material in the biological matrices was demonstrated. Future studies are required to scale up the method and further test blood samples from in vivo trials to completely implement the method. However, here the first steps towards the validation of the method were performed which can help to understand changes in nanoparticles upon exposure to biological fluids in the body. (Less)
Popular Abstract
Tracing nanoparticles in biological matrices

Cancer includes a heterogeneous group of diseases that arises due to an uncontrolled proliferation of cells which are able to invade normal tissues. Magnetic resonance imaging (MRI) has been widely used to detect cancer in its earliest stage. However, there is a need for new contrast agents (CAs) with higher sensitivity. Consequently, cancer nanotechnology has emerged as a new potential field which includes the design of material in a scale range from 1-100 nanometers with clinical use for cancer therapy and diagnosis.

Nanomaterials include a wide range of particles used as versatile molecular devices like vectors for drug delivery or CAs to enhance contrast images from tumor tissues.... (More)
Tracing nanoparticles in biological matrices

Cancer includes a heterogeneous group of diseases that arises due to an uncontrolled proliferation of cells which are able to invade normal tissues. Magnetic resonance imaging (MRI) has been widely used to detect cancer in its earliest stage. However, there is a need for new contrast agents (CAs) with higher sensitivity. Consequently, cancer nanotechnology has emerged as a new potential field which includes the design of material in a scale range from 1-100 nanometers with clinical use for cancer therapy and diagnosis.

Nanomaterials include a wide range of particles used as versatile molecular devices like vectors for drug delivery or CAs to enhance contrast images from tumor tissues. Spago Nanomedical AB in Lund is studying a potential nanomaterial-based contrast agent to be used in MRI. The main structure of the nanoparticle consists of a polymeric central core, which contains a metal ion inside, together with a coating attached to the surface (Figure 1). This coating is made of polyethylene glycol (PEG) chains which form a passive surface that reduces aggregation and undesired interactions in the body.

These particles can be delivered into the tumor tissue through passive targeting. This strategy can be used because during their growth, cancer cells create new but defective blood vessels. Thus, tumor vasculature has an enhanced permeability and lack of an efficient lymphatic drainage which gives rise to the so called enhanced permeability and retention (EPR) effect. This effect allows the nanoparticles to accumulate selectively in the tumor and remain there for a longer time compared to a normal tissue.

Do nanoparticles look the same after being injected into the body?
In this project, a dummy particle mimicking a nanoparticle-based contrast agent was further investigated to set up a combined bioanalysis method to extract and characterize the nanomaterial from relevant biological matrices (e.g urine, serum). The final goal was to demonstrate that the identity of the particles (the main physicochemical properties) remain unaffected after being injected into the blood of animals during in vivo trials. Problems can arise if the content of the nanoparticles is released to the biological fluids or if particles aggregate and lodge somewhere in the body. The nanoparticles were isolated and separated by size using gel permeation chromatography (GPC). The resulting fractions were analyzed in terms of composition and coating using inductively coupled plasma optical emission spectroscopy (ICP-OES) and enzyme-linked immunosorbent assay (ELISA) respectively. The results proved the presence of nanomaterial in the biological matrices and showed a successful separation of the nanoparticles by size with high rate of recovery. Furthermore, it was possible to analyze the core and coating components of the extracted material in an accurate and efficient way. This study shows the importance of characterizing the nanomaterial and proves its identity before going into clinical trials.

Advisor: Dr. Sania Bäckström
Master´s Degree Project in Molecular Biology, 45 credits, 2015
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Fernández, Gema
supervisor
organization
course
MOBN01 20151
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
7409834
date added to LUP
2015-06-22 15:07:30
date last changed
2015-06-22 15:07:30
@misc{7409834,
  abstract     = {Molecular imaging techniques have become an essential tool for cancer diagnosis. They have the potential to detect cancer at early stages and thus, they can change the outcome of the disease and patient prognosis. Magnetic resonance imaging (MRI) has arisen as one of the most promising imaging methods used for the screening of soft tumor tissues such as breast cancer. However, the specificity of MRI remains poor leading to misdiagnosis and many false positive findings. Thus, a wide range of new contrast agents (CAs) are being developed in order to enhance image resolution and safety in cancer diagnosis. For new drugs with an intended clinical use, it is necessary a deep insight into the viability of the molecule prior to clinical trials. Some in vitro analysis and in vivo animal studies are carried out in order to characterize the physicochemical properties of the compound and its toxicity. Problems to find the right methodology to detect and assess identity of nanoparticles after being injected into the blood have been previously found. In this project, a combined method to extract and characterize dummy particles, mimicking nanoparticles to be used as CAs, was developed. These particles consist of a polymeric core, which contains a metal ion inside, together with a coating attached to the surface. A new approach for characterization was investigated by using three analytical techniques: gel permeation chromatography (GPC) to extract and separate the nanoparticles by size, enzyme-linked immunosorbent assay (ELISA) to analyze the coating and inductively coupled plasma optical emission spectroscopy (ICP-OES) to analyze the core and metal ion. Different biological matrices, mainly serum and urine, were tested to prove identity. Results showed an efficient extraction of the material with a high rate of recovery by GPC. Moreover, the accuracy and sensitivity of ELISA and ICP-OES in detecting the coating and composition of nanoparticles respectively was proved. More importantly, identity of the extracted material in the biological matrices was demonstrated. Future studies are required to scale up the method and further test blood samples from in vivo trials to completely implement the method. However, here the first steps towards the validation of the method were performed which can help to understand changes in nanoparticles upon exposure to biological fluids in the body.},
  author       = {Fernández, Gema},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of a method for bioanalysis of a nanomaterial in biological matrices},
  year         = {2015},
}