The Design of Hybrid Nanoparticles for Image ... - ACS Publications

Chapter 7

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The Design of Hybrid Nanoparticles for Image-Guided Radiotherapy Christophe Alric,a Rana Bazzi,b François Lux,a Gautier Laurent,b Matteo Martini,a Marie Dutreix,c Géraldine Le Duc,d Pascal Perriat,e Stéphane Roux,*,b and Olivier Tillementa aLaboratoire

de Physico-Chimie des Matériaux Luminescents, UMR 5620 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France bInstitut UTINAM, UMR 6213 CNRS - Université de Franche-Comté, 16 route de Gray, 25030 Besançon, France cInstitut Curie, Equipe, “Recombinaison et instabilité des génomes”, UMR 2027, Orsay, France dEuropean Synchrotron Radiation Facility, ID 17 Biomedical Beamline, Polygone Scientifique Louis Néel, 6 rue Jules Horowitz, 38000 Grenoble, France eMatériaux Ingénierie et Science, UMR 5510 CNRS, INSA de Lyon, 69621 Villeurbanne Cedex, France *E-mail: [email protected]

Many studies revealed the high potential of multifunctional nanoparticles for biomedical applications. Since these nanoparticles can be designed for combining imaging and remotely controlled therapeutic activity, image-guided therapy which rests on the induction of nanoparticles toxicity by external stimulus when the nanoparticles content is both high in the diseased zone and low in the healthy tissue can be envisaged, especially for fighting cancer (one of the most important cause of mortality in several countries). Image-guided therapy should lead to valuable improvements in radiation-based therapy provided that the multifunctional radiosensitizing nanoparticles developed for increasing the selectivity of the radiotherapy (and therefore the efficiency) meet the criteria imposed by in vivo applications and by the physical principles of the interaction between radiation and the matter. © 2012 American Chemical Society In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Diagnosis and Treatment of Cancer


Cancer The term cancer includes a set of diseases characterized by unlimited proliferation of cells that, following the alteration of their genetic heritage, escape the normal mechanisms of differentiation and regulation of their proliferation. This increase in surplus populations of cells destroys the surrounding tissue and may be accompanied by a dispersion throughout the body. In many cases, this can cause death (1). There are many different types of cancer (more than a hundred) that can affect any part of the body. The most common cancers in women are (in descending order of frequency) breast cancer, colorectal cancer and cancers of the cervix and lungs. In men, lung cancer and prostate cancer are most common, followed by colorectal and stomach cancer (2). An estimated eleven million cancers are diagnosed worldwide each year and between seven and nine million people die, which is about 13% of global mortality. According to estimates by international health agencies, the annual number of cancer deaths is expected to rise to twelve million in 2030 (3).

Induction and Growth It is now accepted that the development of cancer can be outlined in three phases, which can take several years in total (1):

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The initiation, which corresponds to irreversible damage to the DNA of a cell (either spontaneously or after exposure to a chemical, physical, or biological carcinogen...). The “initiated” cell multiplies; The promotion, which corresponds to prolonged exposure (repeated or continuous) to a substance that maintains and stabilizes the lesion through the proliferation of “initiated” cells; The progression, which corresponds to the acquisition of the properties of uncontrolled proliferation, acquisition of independence, loss of differentiation, local and metastatic invasion.

Cancer cells are fundamentally different from “normal” cells. They have the ability to escape the regulatory mechanisms of the cell population (homeostasis) and thus gain "immortality" allowing them to divide again and again (1, 4). Moreover, they can implement various mechanisms to avoid detection or destruction by the immune system (tumor escape) (5). This allows them to multiply rapidly and without any control. Growth of a malignant tumor is then very fast and leads to the invasion and destruction of surrounding healthy tissue (tumor invasion). Through the secretion of specific enzymes, tumor cells degrade the extracellular matrix, making it more loose and disorganized. Loss of adhesion between the cells and the displacement 96 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

acquisition capacity (cell migration) result in the access of tumor cells to vessels (1, 4). The latter are of fundamental importance for tumor growth since they ensure the delivery of nutriments and oxygen to the tumor.


Angiogenesis and Metastasis

In normal physiological conditions, the creation of new blood capillaries from preexisting vessels (angiogenesis) is finely controlled by complex molecular mechanisms (balance between induction and inhibition of the angiogenesis) by taking into account the physiological needs (6). Certain stimuli (hypoxia, pressure generated by a large proliferation of cells) can lead to a shift in the balance regulating the formation of new blood vessels. This change, designated as the “angiogenic switch”, is reflected by the increase in the concentration of angiogenesis inducing molecules which is concomitant with the decrease in the concentration of inhibiting molecules (7). Up to a volume of about 1 mm3, oxygen and nutrients are delivered to a growing solid tumor by diffusion through the surrounding interstitial spaces. Beyond this critical size, the tumor is exposed to hypoxia (8). Cancer cells secrete many factors inducing the angiogenic switch. As a result, blood vessels are created from preexisting vasculature (9, 10). These factors, such as vascular endothelial growth factor (VEGF) or fibroblast growth factors (FGFs) are involved in proliferation and arrangement of endothelial cells necessary for the formation of new vessels which will then bring oxygen and nutrients for tumor growth. The latter begins to grow exponentially. The formation of lymphatic neo-vessels was also initiated by the secretion of growth factors in tumor development (11). The formation of new vessels (blood and lymph) induced by the growth of a solid tumor appears to be the essential phenomenon for the dissemination of cancer cells in the body (11, 12). They are able to break into new vessels (intravasion), to travel and then to extract (extravasation) to reach another place. Cancer cells can then establish distant colonies (metastases). They may then induce neovascularization within their location in order to grow and spread in the body (13, 14). Metastases are by far the largest cause of mortality from cancer (15). The establishment of neovascularization is a sequenced process essential for tumor growth and metastasis (10, 16). The resulting new vessels which are made haphazardly, constitute a prime target for many innovative treatments (17, 18).

EPR Effect

Solid tumors generally have a different physiology from that of healthy tissue (19, 20). These different specificities, which depend on the types of cancers, can be exploited in order to only treat tumors while sparing the surrounding healthy tissue. 97 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


One of the most remarkable illustrations of the physiology of solid tumors is their hyper-vascularization, which is structurally and functionally abnormal in most tumors (7, 21). Because of their rapid growth which is continually induced by growth factors, newly formed blood vessels are often disorganized, tortuous and have non-constant diameter (7, 22, 23). Endothelial cells that compose them are often not contiguous. The fenestration of the vessels which irrigate the tumors is characterized by void spaces between cells of hundreds of nanometers (24–26), whereas in the case of continuous healthy endothelium of blood capillaries, the distance between endothelial cells is of the order of 3 to 6 nm, allowing the passage of ions and small molecules necessary for the microcirculation (27) (Figure 1).

Figure 1. Schematic representation of blood vessels irrigating healthy tissue (left) and tumor (right). (see color insert) Blood vessels from neoangiogenesis are often porous and permeable (28, 29). Another notable feature of the environment of solid tumors is the absence of fully functional lymphatic vessels, despite the lymphogenesis initiated by growth factors (7, 30). Associated with permeable hyper-vascularization of tumor, the lack of lymphatic drainage may result in accumulation of macromolecules in the interstitial spaces, that can be exploited to target the tumor environment (31). This phenomenon, known as the EPR effect (Enhanced Permeability and Retention Effect), allows a "passive" targeting of highly vascularized solid tumors, using macromolecules or nano-objects (Figure 1). The EPR effect is now one of the major pathways for the detection and treatment of solid tumors (32). Moreover in the case of solid tumors, most of the cells (both cancerous and stroma cells) overexpress on their surface, because of extensive metabolism, different molecules which are less present on normal cells. These molecules 98 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


are mostly membrane receptors (proteins) involved in the growth and survival of cancer cells. Their expression differs among cancer types. For example, receptors for growth factors, such as EGFR (Epidermal Growth Factor Receptor), are overexpressed in some cancers including colorectal or HER2 (Human Epidermal Growth Factor Receptor-2), from certain types of breast cancer. The overexpression of these markers gives sometimes an estimation of the virulence of the disease and permits to assess the prognosis (33, 34). Many ligands have been developed to allow specific targeting of cancerous cell surface receptors, thus paving the way for targeted therapies (35–37) (Figure 1). In most cases, active targeting is possible only if passive targeting is observed. The attractive potential of active targeting does not rest only on the increase of accumulated therapeutic agents in the tumor but also in the increase of the residence time owing to the interaction with cancerous cell receptors. The return in bloodstream of the therapeutic agents occurs indeed more belatedly than in the case of passive targeting. Another promising strategy is the targeting of receptors expressed by stroma cells and tumor vascularization, particularly the neoangiogenic endothelial cells (38, 39). The vascularization plays an important nutritional role in the survival and growth of solid tumors (intake of oxygen and nutrients) but also allows its metastasis. It is therefore an essential target in anticancer strategy (40, 41). The inhibition of neoangiogenesis or the selective destruction of new vessels intended to indirectly causes the death of cancerous cells by depriving them of nutrient and oxygen they need to live and multiply. The antiangiogenic and antivascular therapies are based on the overexpression of membrane markers on the neoangiogenic endothelial cells (as compared to endothelial cells of healthy tissues). Solid tumors have remarkable physiological characteristics (permeable and uncontrolled vascularization, overexpression of membrane receptors by tumor cells or stroma cells) for different types of cancers and can evolve during their progression. These features can be exploited to detect as early as possible and treat selectively the tumor areas. Currently, medical imaging remains one of the major tools for cancer detection (1, 42).

Medical Imaging for the Visualization of Solid Tumors

Medical imaging is now one of the pillars of the fight against cancer. Its contribution in this field includes the detection of lesions before the onset of clinical signs, the evaluation of a stage of disease progression and of the response to treatment and/or the planning of a therapeutic protocol. Medical imaging plays an important role in defining target volumes for radiation treatment (43, 44). Born at the end of 19th century with the discovery and use of X-rays to visualize bone structures, medical imaging today consists of several techniques for noninvasively visualizing within a living organism. These techniques rely on different physical principles that determine their resolution, sensitivity and type of tissue or biological phenomena that they render visible (45, 46). 99 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


X-ray Imaging

The X-ray imaging is an indispensable technique in medical imaging. Since the discovery of X-rays by Roentgen in 1895, it was the only imaging technique available until the mid-twentieth century. The progress in computer science contributed to the improvements of X-ray imaging. The X-ray imaging is based on the differential attenuation of a beam of incident X-rays (25–150 keV) by the body tissues. A X-ray beam passes through a patient and is collected on a photographic plate or a photon detector before being converted into digital signals. Tissues absorb radiation depending on their composition (atomic number and density) and thickness. The invention of X-ray computed tomography (or “CT scanner”) by Hounsfield in 1972 revolutionized the X-ray imaging. It is based on the acquisition of multiple projections of the patient at different angles through a system of rotation of the X-ray tube (47). CT scanner quickly provides three-dimensional snapshots of the living organism anatomy, with excellent spatial resolution (~ 50 microns). This technique is also able to image in a single session deep tissues (whole body). X-ray computed tomography is now one of the major tools in medical imaging (48). The main limitation of the X-ray imaging is the use of ionizing radiation, which limits the number of examinations per patient (48). This technique is also hampered by a poor resolution of soft tissue and a low sensitivity (42). The injection of contrast agents allows in some extent improving the contrast of soft tissues, but the sensitivity of the X-ray imaging remains below that of other imaging techniques. The majority of contrast agents for X-ray imaging are iodinated molecules. Iodine increases the contrast of soft tissues (where contrast agents are) by the photoelectric effect, thanks to its high atomic number (Z(I) = 53). The contrast agents for X-ray imaging are derivatives of 1,3,5-triiodobenzene (49). Once injected into the bloodstream, iodinated contrast agents (ICAs) are spread throughout the extracellular space due to their low molecular weight (less than 2000 g.mol-1) and are rapidly cleared from the body, mainly by renal excretion (50). They allow both better visualization of soft tissue and a transient contrast enhancement between normal tissue and diseased tissues (including tumor hypervascularization). The limitations of molecular ICAs are their rapid elimination from the body, their non-specific distribution and high viscosity due to very high concentrations of iodine (from 100 to 370 g.L–1).

Magnetic Resonance Imaging (MRI)

The magnetic resonance imaging (MRI) is a medical imaging technique which provides highly resolved three-dimensional images of living bodies using nuclear magnetic resonance (NMR). MRI exploits the magnetic properties of protons (constituting about 85% of biological tissue) to image inside a body, without using ionizing radiation (51). Parameters at the origin of contrast in MRI images are the relaxation time of the tissue magnetization (T1 and T2) 100 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


and the density of protons ρ in these tissues. The relaxation times are identical for the same type of tissue and therefore allow to distinguish different tissue types. The relaxation times are sensitive to the physicochemical environment of protons in imaged tissues. MRI allows obtaining anatomical images of soft tissue (containing many protons) of very good quality and is not limited in depth acquisition. It is the reference technique for the study of central nervous system, both for anatomical and functional study (e.g. brain). MRI is currently one of the most commonly used imaging techniques, especially for the detection of tumors. However, MRI has significant limitations, which include a long acquisition time and the inability to image large areas (e.g. the whole body) in a single acquisition. Another weakness of this technique is its low sensitivity which sometimes makes difficult the distinction between normal and pathological tissues. For more reliable medical diagnosis, the administration of contrast agents to patients is commonly used to enhance the native contrast between different tissues. The use of magnetic contrast agents can enhance the natural contrast between different tissue types in decreasing the relaxation time of protons they contain. The ability of a contrast agent to modify the relaxation time of protons that surround it is quantitatively represented by its relaxivity (longitudinal r1 or transversal r2 relaxivity). The contrast agents for MRI are divided into two types: superparamagnetic agents and paramagnetic compounds (52). Even if they act on both longitudinal (T1) and transverse (T2) relaxation times of protons, their effect is only predominant on one out of two. The superparamagnetic agents (T2 or negative contrast agents) are iron oxide nanoparticles (5 to 200 nm) coated with a layer of hydrophilic polymer (e.g. dextran). Depending on their size, they are called SPIOs (SuperParamagnetic Iron Oxides, > 50 nm) or USPIOs (Ultrasmall SuperParamagnetic Iron Oxides,

The Design of Hybrid Nanoparticles for Image ... - ACS Publications

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