Advanced Theranostic Materials

By Ashutosh Tiwari

The present book is covers the recent advances in the development on the regulation of such theragnosis system and their biomedical perspectives to act as a future nanomedicine. Advanced Theranostics Materialsis written by a distinguished group of contributors and provides comprehensive coverage of the current literature, up-to-date overview of all aspects of advanced theranostics materials ranging from system biology, diagnostics, imaging, image-guided therapy, therapeutics, biosensors, and translational medicine and personalized medicine, as well as the much broader task of covering most topics of biomedical research. The books focusses on the following topics:

Part 1: System biology and translational medicine

• Aberrant Signaling Pathways: Hallmark of Cancer Cells and Target for Nanotherapeutics
• Application of Nanoparticles in Cancer Treatment
• Biomacromolecule-Gated Mesoporous Silica Drug Delivery Systems
• Construction of Functional DNA Nanostructures for Theranostic Applications
• Smart Polypeptide Nanocarriers for Malignancy Therapeutics

Part 2: Imaging and therapeutics

• Dimercaptosuccinic acid-coated magnetic nanoparticles as a localized delivery system in cancer immunotherapy
• Cardiovascular nanomedicine
• Chitosan-based systems for sustained drug release
• Nanocapsules in biomedicine: promises and challenges
• Chitosan-based polyelectrolyte complexes: characteristics and application in formulation of particulate drug carriers

Part 3: Diagnostics and featured prognostics

• Non-invasive Glucose Biosensors based on Nanomaterials
• Self/directed Assembly of Nanoparticles: A review on various approaches
• Ion exchangers – an open window for the development of advanced materials with pharmaceutical and medical applications

New Titanium Alloys for Biomedical Applications

• Language: English
• Publisher: Wiley
• Release date: Jun 30, 2015
• ISBN: 9781118998915

Book preview

Preface

Since ancient times, the advancement of human civilization has always been driven by smart engineering. Counted among the ancient professions, technicians still continue to be a compelling force for advancing civilization. However, advancement always brings new challenges followed by smarter solutions followed by more challenges. Scientists and doctors have created outstanding advancements in medicine, but those improvements have also posed several obstacles. Biochemists, who for centuries have been leaders in the biomedical research field, are facing newfangled challenges in developing medicine, along with suggestions for potential solutions. Since the future of medicine is going to be personalized, bioengineers are are working very hard in a cross-disciplinary manner to find a way to craft auspicious personalized drugs.

The most promising approach for individualized medicine is known as ‘theranostics,’ a term coined in 2002 which is defined as a blend of therapeutics and diagnostics. The development of a theranostic approach would not only guide the clinician in prescribing the appropriate drugs to patients, but would also ensure spatio-temporal distribution of drugs within the patient’s body in order to ensure a safe and healthy recovery. Though a lot of research and development still needs to be done before theranostics becomes a reality, the potential outcome of the use of this approach is incredible, constructive, and ethical.

This book strives to accumulate current advances in the design and optimization of biocompatible material and technologies by formulating novel and smart ‘theranostic’ modules for next-generation applications. Theranostics itself covers the cross-disciplinary fields of chemistry, biology, materials and engineering and plays a vital role in medical science and technology. Over the entire past decade, an enormous assortment of theranostic modules have been formulated and optimized for potential clinical trial. ‘Advanced Theranostics Materials’ offers comprehensive chapters on the current status of concrete approaches and their potential range of applications. The concept of design with precise functions and effective self-reporting is of great interest and has massive prospective application in personalized medicine.

Written by an eminent group of scientists, this book is appropriate for a wide spectrum of readers from diverse backgrounds, including clinicians, and would be of great interest for experts in both academia and industry. It not only provides readers with a comprehensive exposure to the current status, but also gives up-to-date approaches for all aspects of advanced theranostic materials. For those who want to start working in related fields, the required fundamental background necessary for future design and optimization is presented, though the scope of this book is far broader and focuses on the emergent area of theranostics and their applications.

The editors dedicate this first-ever book on ‘Advanced Theranostics’ to their respective grandparents (Tiwari and Patra) who passed away during the editing of this book cognizant with the belief that departing souls always bring incredible blessings to the newcomers such as ‘theranostics’.

The Editors

Ashutosh Tiwari, PhD, DSc

Hirak K Patra, PhD

Jeong-Woo Choi, PhD

Part 1

SYSTEM BIOLOGY AND TRANSLATIONAL MEDICINE

Chapter 1

Aberrant Signaling Pathways

Hallmark of Cancer Cells and Target for Nanotherapeutics

Gulnaz T. Javan¹, Sheree J. Finley², Ismail Can¹, Amandeep Salhotra³, Ashim Malhotra⁴, and Shivani Soni⁵,*

¹Forensic Science Program, Physical Sciences Department, Alabama State University, Montgomery, AL, USA

²Physical Sciences Department, Alabama State University, Montgomery, AL, USA

³Department of Hematology & Hematopoietic Cell Transplantation, City of Hope, Duarte, CA, USA

⁴Department of Pharmaceutical Sciences, School of Pharmacy, Pacific University, Hillsboro, OR, USA

⁵Department of Biological Sciences, Alabama State University, Montgomery, AL, USA

*Corresponding author: [e-mail]

Abstract

Nanotechnology is the field that applies to the design, engineering, and validation of manufacture processes and application of entities that exist at the scale of 1 × 10−9 m. The advent of this technology has revolutionized both basic and applied sciences. One prime usage of this technology that has recently emerged is in the field of medicine, specifically in diagnostic, interventional, pharmacological, and formulation sciences as applicable to the treatment of cancers. Interestingly, nanotechnology and cancer research and treatment are naturally consanguineous due to multiple challenges that are posed by cancer and the solutions that are readily offered by nanotechnology. This chapter seeks to address the use of advanced materials for the construction of nanoscale vectors that simultaneously addresses the issues of neoplastic therapy. We shall discuss the problem of scale, with particular reference to the recently unraveled molecular biology of cancer and how nanotechnology provides the adequate medium to intervene and inject pharmacological molecules at this intracellular dimension. The use of nanoparticles in targeting specific receptors such as the protein tyrosine kinases or nonfunctioning second messenger systems downstream of these kinases such as the ras-rak system, which become aberrant in cancer, will be discussed. The emerging roles of nanotechnology-based targeted delivery, the manufacture of nanoscale controlled release formulations, and the effects of advanced materials used for the construction of nano-vehicles on pharmaceutical formulation and stability will be discussed.

1.1 Cancer

Cancer is clonal spread of cells with impaired growth characteristics [1]. It is the most complicated and most common of the somatic genetic diseases. Cancer is blamed on multi-factors which include bacteria, virus, inheritance, chemicals, radiation, diet, environment, and life style to name a few [2–5].

Cancer is a costly and fatal disease. In 2012, 56 million people died worldwide out of which 8.2 million died from cancer. About 30% of cancers are curable and statics show that 60% of new cancer cases occur in Africa, Asia, and central and South America. Non-communicable diseases accounted for more than 68% of all deaths globally in 2012. The top four non-communicable diseases include cancers, chronic lung diseases, cardiovascular diseases, and diabetes.

1.2 Pathways Deregulated in Cancer: Introduction

Cancer is a genetically complex, fatal, and hard to treat disease. It is the second leading cause of death which causes an average of 1500 deaths a day in United States [6]. Despite recent advances in understanding of the fundamentals of cancer, advances in clinical treatment of cancer and successful prevention are far too few. Some reasons for this lack of advancement in treatment include toxic effects of chemotherapy agents on healthy cells and rapid acquisition of resistance against these treatments by cancerous cells [6]. Introduction of nanotechnology to medicine has opened up an array of different approaches to cancer treatment and target poisoning of the cancer cells. This new approach enables scientists to use nanoscaled structures to carry payloads of chemotherapeutic agents, diagnostic chemical, or imaging agents to site of cancer and conduct precise intervention. Advances in nanotechnology are making target treatment of cancer cells a reality; however, the challenge of overcoming cells resistance to treatment is still a more important goal to achieve. To understand and appreciate the complexity of cancer and how a normal cell turns into a cancerous cell, one must understand normal cell growth as well as all pathways and signals that lead to cell growth, division, metabolism, and survival. It is also as important to use correct molecular classification, instead of traditional methods of using morphological appearance to classify cancer to streamline treatment. Gene expression can be used for class prediction and class discovery [7]. Moreover, miRNA which has a role in almost all aspects of cell behavior is the major regulator of gene expression. This is why miRNA is the target of different studies to discover extent of its involvement in cancer and its role as oncogene or tumor suppressor gene (TSG) [8]. Normal cell growth and division are controlled by many signals and follows specific pathways. Any damage to pathways or signaling system which results in abnormal cell growth or division is generally corrected by apoptosis; however cancer cells do not follow or resist this complex cell division and growth. Discovery of proto-oncogenes and TSGs has changed over ever-expanding understanding of cancer cells. Proto-oncogenes influence cell proliferation and extend their survival rate whereas TSGs keep cell growth in check and prevent or inhibit their growth and survival. This disruption of normal cell development by proto-oncogenes causes growth rate of cells to increase and exceed normal cell ability to keep equilibrium between growth and survival [9]. Deregulation of gene expression is another distinguishing property of cancer cells. This deregulation in expression of gene happens due to modifications of DNA’s nucleotides [6]. Douglas Hanahan and Robert Weinberg [10–11] two cancer researchers have simplified complexity of the cancer into a number of underlying principles. They list hallmarks of the cancer cells as (i) self-stimulated cell growth, (ii) resistance against cell’s inhibitory signals, (iii) resistance against apoptosis, (iv) capable of angiogenesis, (v) endless multiplication, (vi) capable of metastasis, (vii) abnormal metabolic pathways, and (viii) eluding immune system. Normal cell growth and division controlled by mechanisms that are governed by several proteins. When a critical protein is damaged due to acquired or somatic mutations, and cell apoptosis does not happen, cancerous cell will result which must have the above mentioned hallmarks in order to multiply and grow.

Cell growth and proliferation are regulated by extracellular signals. Homeostatic regulation is disturbed by oncogenes in cancerous cells which allow them to have self-proliferation capability. Invasion and metastasis of cancer cells are aided by growth factors signaling pathways (Figure 1.1).

Figure 1.1 A subway map of cancer pathways [12].

1.3 Introduction to Nanotechnology

Nanotechnology can be defined as an ever-expanding area of science that focuses on the tiniest particles of structures. This area of science refers to the fabrication, validation and application of manufactured objects and particles on a scale of 1 × 10−9 m, or more specifically, 1 nanometer (nm). Certain characteristics such as fluorescence, cellular permeability and chemical reactivity depend on the size of the nanoparticle. The dimensions of nanoparticulates are constructed on a scale that is 100–1000 times smaller than typical eukaryotic cancer cells [13]. Therefore, nanoparticles provide extremely suitable platforms to construct nano-sized drug delivery systems for neoplastic research and therapy.

Nanotechnology is a multidisciplinary field that synergistically pools the expertise of synthetic chemists, engineers, biophysicists, biologists, and medical research personnel to develop state-of-the-art, nano-sized clinical strategies [14]. The National Nanotechnology Initiative (NNI) was initiated in 2001 as a multi-office agency tasked with oversight of the research and development of world-class investigations of nanoparticles. NNI set initial definitions of nanotechnology at a limit of 100 nm [15]. However, unique physiochemical characteristics emerge that frequently create dimensions greater than 1000 nm [16]. Compared to standard materials, nanostructures are far stronger, more stable, and have a larger surface area per unit [17]. Of note, nanostructures begin exhibiting quantum effects below 10 nm [18].

Examples of organic materials used to manufacture nanomaterials include carbon-based dendrimers, carbon nanorods, carbon nanotubes, fullerenes, and graphenes [19–21]. Inorganic nanoparticles include silica-based glass quantum dots, titanium oxides, and gadolinium nitride nanowires, and metallic-based silver, gold, platinum, and magnetic nanoparticles [19, 21]. The size of the particles ranges from the carbon C60 fullerenes and nanotubes with diameters of approximately 1 nm to liposomes and iron oxide nanoparticles as large as 1000 nm [16] (Figure 1.2)

Figure 1.2 Size comparison of selected nanoparticles and biological matter.

The first report of a nanoparticle platform for medical applications was in 1965 with the creation of spontaneously formed aqueous suspensions of phospholipid liquid crystals that appeared to bind or capture metal cations [22]. Within the past two decades, robust academic and industrial investigations in nanotechnology have emerged resulting in many different fields of clinical application. Thus, the diverse arrays of applications of nanoparticles as cell-specific, anticancer vehicles are poised to build a new generation of innovative tools to treat cancer.

The research and development of nanotechnology has experienced quasi-exponential growth as evidenced by the proliferation of patents and research articles published in scientific literature over the last 20 years. As of 2013, more than 500,000 research articles have been published on the topic of nanotechnology [23]. According to PubMed, only four research articles were published on the topic of nanoparticles in the 1970s, but the number grew to 15,631 in 2013 (Figure 1.3a and b). Specifically in the field of nanomedicine, more than 70% of scientific papers and nearly 60% of all patents filed relate to drug delivery applications [21, 24].

Figure 1.3 (a) The number of nanoparticle research articles published between 1978 and 2013 according to PubMed.gov [25]. (b) The number of nanoparticle research articles published between 1978 and 2013 according to PubMed.gov [25].

1.3.1 Overview of Clinical Nanotechnology

Nanoparticles act as biological transport vehicles that facilitate the dispersion of hydrophobic drugs that are otherwise insoluble in aqueous solutions like blood. Encapsulated drugs are released over a period of time from the nanocarriers in a controlled manner that maintains drug concentrations at sustained efficacy while avoiding harm to healthy cells. The release of the drug is activated by a stimulus unique to the delivery site.

The progression of clinical nanotechnology as it relates to cancers has produced customized synthetic drug delivery systems of nanoscale proportions to deliver chemotherapeutic adjuvants and imaging agents to targeted cancer cells. As of 2012, the US Federal Drug Administration (FDA) had approved 247 nanomedicine agents (drugs, imaging molecules, and vaccines) for different stages of preclinical and clinical study [15, 21, 26]. The use of imaging nanotechnology has produced visualizing agents that enable early detection of diseased cells. Together, these uses of nanoparticles have revolutionized the field of cancer therapeutics. Most importantly, the use of imaging nanotechnology is extremely beneficial to the treatment of cancer because it enables early detection which leads to significant reductions in the cancer mortality rates [27].

Incidences and deaths by cancers are pervasive public health problems in the Unites States and globally. Although the incidences of cancer and cancer-related deaths in the United States has declined over the last decade, the death rate still remains at almost 1600 deaths per day [27]. In the United States, the National Cancer Institute (NCI), the Centers for Disease Control and Prevention, the North American Association of Central Cancer Registries, and the National Center of Health Statistics collectively project that the number of cancer deaths will surpass 585,000 in 2014 [28]. Globally, according to the World Health Organization (WHO), cancers are the most prevalent causes of deaths worldwide [29]. The 2012 statistics provided by WHO stated that the number of mortalities due to cancers was 8.2 million [29].

Conventional cancer treatments act to interfere with DNA processes prompting the induction of cell cycle arrest that ultimately induces apoptosis in highly proliferating neoplastic cells, and unfortunately, in healthy cells as well. Thus, nanomedicine offers significant advantages to traditional chemotherapeutic treatments. The primary advantage is the fact that nanoparticles can be medically engineered and constructed such that their therapeutics can be customized to be recognized by specific targets, i.e., cancerous cells. Another major advantage is that nanoparticulate drug delivery systems can administer the adjuvants via oral [30], nasal [31], parenteral [32], and intra-ocular routes [33].

Traditionally, cancer therapies were limited to surgery, followed by long courses of chemotherapy or radiation and, commonly a combination of these two [13, 27]. It is reported that residual cancer cells are frequently left behind after surgical removal of the tumor [27]. Other disadvantages include the nonspecific biodistribution of traditional chemotherapeutic agents to not only diseased cells, but also to noncancerous cells [34]. This phenomenon leads to excessive cytotoxicity. The nonspecificity thus limits the dosage allowances of the chemotherapeutic agents which influence inadequate drug concentrations reaching the diseased cells [13, 34]. Another disadvantage to conventional therapies involves the restricted capacity to monitor the treatment responses to the therapies [13].

1.3.1.1 Physicochemical Structure of Nanoparticles

Generally, the physicochemical structure of nanoparticles includes the following four components: internal core, payload, corona and targeting ligand [35]. The internal core contains the hydrophobic payload – the chemotherapeutic adjuvant or imaging agent. A hydrophilic, proteinaceous corona forms the protective coating that surrounds and stabilizes the core. The corona interacts with biological factors, such as relatively small proteins, receptors, and antibodies [36]. By interacting with these factors, it plays a crucial role of the biodistribution and dissemination of the nanoparticle throughout body [37]. Site-specific targeting ligands are functionalized onto the corona to facilitate the internalization of the nanoparticle. As the nanoparticle accumulates in tumor tissue, the targeting ligand binds to receptors on the cell surfaces. Hence, these site-specific internalizing ligands affect the cellular uptake by increasing binding and facilitating endocytosis of the particle. Cell penetrating peptides are short peptides with transduction domains and have been shown to achieve up to a 100-fold more transfection compared to equivalent nanoparticles with no such cell penetrating peptide [50]. For example, polycation ligands, such as surface phosphonate groups functionalized with branched polyethylenimine enhances cellular uptake of silica nanoparticles [38]. Monoclonal antibodies and antibody fragments are commonly used as ligands and have high binding ability [13].

1.3.1.2 Drug Delivery Strategies

The general method of drug delivery systems is as follows: the nanoparticle-coupled tumor-specific target ligand binds to the surface receptors of the tumor [13]. This binding initiates receptor-mediated endocytosis through an endosome-dependent membrane invagination [48]. As the interior of the endosome becomes increasingly acidic, drugs are released from the nanoparticle in to the cytoplasm of the tumor cell. Internalization of nanoparticles inside cells is achieved through a variety of delivery approaches.

There are two major strategies employed by advanced drug delivery systems for the uptake of the nanocarriers in situ. Passive targeting, also known as the enhanced permeation and retention (EPR) effect, targets cancer by increased drug accumulation and entrapment of nanoparticles in tumors [39]. The phenomenon is accomplished due to characteristic dysfunctional, leaky angiogenetic vessels and poor lymphatic drainage [21]. The extensive network of leaky vessel structure occurs in higher proportions in cancerous tissues compared to normal [39]. The nanocarriers leak through fenestrae in the vessels that can be up to 600 nm in size [40].

The other targeting system is active targeting. Active targeting exploits the fact that there is well-documented overexpression of receptors on the surfaces of tumor cells [41]. This phenomenon is a characteristic structural and biochemical feature of tumors and it differentiates diseased cells from noncancerous cells. Therefore, in principle, the targeting ligands are designed to preferentially recognize the overexpression of receptors on the periphery of cancer cells while also exhibiting limited recognition of the normally expressed receptors on healthy cells.

Another well-reported targeting strategy exploits the fact that metabolic activity of tumor cells typically results in the accumulation of acidic by-products in the extracellular microenvironment surrounding the cells [36]. The production of lactic acid under anaerobic conditions and the hydrolysis of ATP in the tumor cells’ energy-deficient environment contribute to the acrid microenvironment of tumor cells [42].

During endocytosis of nanoparticulate drugs, targeting ligands fuse with the cell membrane which allows the escape of the adjuvants into the cytoplasm of the cell to deliver the therapeutic payload [35]. Once in the cell, the hydrophobic drug must overcome the barriers created by the corona. The drugs are engineered to promote release of the drug from the manufactured inclusions in a method known as endosomal escape [21]. While it is well documented that the extracellular microenvironment is acidic, on the contrary, intracellular pH is more basic in tumor cells compared to normal cells [43]. The observed differences provide opportunity for exploitable avenues for the treatment of cancer. Weakly acidic, lipid-soluble drugs diffuse freely across the tumor cell membrane. In the relatively basic cytoplasm, the drugs accumulate. Hence, the substantial pH differences in the intracellular and extracellular conditions of tumor versus noncancerous cells provide means for the drug delivery systems to advantageously exploit the environments.

Another important factor in the advanced drug delivery systems is the degradation of the drug by lysosomal enzymes. A drug delivery system must accomplish a balance between bioavailability while limiting its degradation. Studies have demonstrated the escape of biodegradable nanoparticles, such as those formulated using poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles, permit the rapid escape from the endo-lysosomal compartment into the cytosol following their uptake [44].

Several designs are used to couple the drug into its nano-sized delivery vehicle. Most assembly methods either entrap the drug inside internal core made of liposomes, mesoporous nanoparticles or some other hollow nanoparticles, or alternatively, attach the drug covalently to the surface of nanoparticles [45]. Several nanoparticulate fabrications involve self-assembly, methods in which the components of the nanostructure assemble spontaneously through intermolecular interactions. The assemblages start with solubilized small-molecule precursors such as gold nanoparticles or lipid structures, and they form higher-ordered structures with internal cores that are suitable for encapsulation of chemotherapeutic agents [46]. The typical diameter of self-assembly encapsulation is 100–200 nm [47].

Nanoprecipitation [48] and nanoemulsion–diffusion [49] are also used to fabricate nanoparticles, but they are used in fewer applications. Nanoprecipitation technology is also known as interfacial deposition or solvent displacement [50]. Nanoprecipitation involves the formation of polymers through dropwise or moderately stirred polymer dissolved in an organic solvent is added to water in droplets. Nanoparticles form instantly by solvent diffusion. Nanoemulsion technology involves the use of nontoxic oils that are emulsified with surfactants using a high-shear homogenizer to stabilize the emulsion. When the nanoparticles encounter lipids on the cell membrane, the lipids merge initiating endocytosis.

Another important factor in the advanced drug delivery systems is the degradation of the drug by lysosomal enzymes. A drug delivery system must accomplish a balance between bioavailability while limiting its degradation.

1.3.2 Current Usage in Cancer Treatment

In the 1980s, the first generation of biologically therapeutic nanoparticles that had wide applications in cancer treatments were drugs encapsulated through hydrophobic, lipid-based interactions [19, 20, 21, 27, 46]. The leaders in nanocarrier development are liposomal formulations and polymeric-based nanoparticles [21]. Liposomal formulations spontaneously arise in situ when noncovalent hydrophobic interactions assemble phospholipid systems into lamellar phase bilayers. Liposomes have the advantage of being biocompatible which reduces the side effects without diminishing the effectiveness of the drug [51]. However, its high cost of fabrication, poor shelf life, and slow release of the drug payload are some of their disadvantages. It is also suitable for a variety of administration routes. Studies have shown that liposomes that have diameters of 150–200 nm remain in the bloodstream longer than those with smaller diameters that are less than 70 nm [15].

As of 2012, the US FDA had approved 247 nanomedicine agents (drugs and imaging molecules) for different stages of preclinical and clinical study [15, 21]. One major advantage of nanomedicines is the fact drug bioavailability is greater for nanotherapeutics because of their relatively high surface area [18]. They also have an enhanced ability to transporting relatively large doses of toxic, therapeutic cocktails specifically to the diseased cells. Additionally, their inherent design allows gradual release the drug after delivery, which is critical for an effective cancer treatment.

As stated previously, the predominant groups of nanotherapeutics are liposomal and polymeric drugs. Spherical liposomes have garnered the most success as drug carriers with greater than 10 drug formulations approved by the US FDA and other similar regulatory administrations with several others presently in clinical trials [46].

PEGylated, liposomal-encapsulated formulation of doxorubicin (Doxil®), in a combination of both liposomal and polymeric nanotherapeutics, were the first to be approved by the US FDA for the passive targeting treatment of Kaposi’s sarcoma, refractory ovarian cancers and metastatic breast cancer [52, 27]. Myocet™ is liposomal formulstion of doxorubicin approved for the treatment of metastatic breast cancer [21]. Studies have shown that doxorubicin-encapsulated liposomes have bioavailability 87-fold higher than free doxorubicin 7 days after injection [52]. Daunoxome™ is another liposomal formulation of daunorubicin approved for the treatment of Kaposi’s sarcoma [21].

Of the polymeric nanomedicines, as of 2014 three polymer protein, passive targeting platforms have been approved by the US FDA. Zinostatin Stimaler® is polymer protein formulation of neocarzinostatin with styrene maleic acid anhydride copolymer (SMANCS) approved for the treatment of hepatocellular carcinoma [21]. Oncospar® is a PEG-l-asparagine formulation approved for the treatment of acute lymphoblastic leukemia [21]. Neulasta® is another polymeric construct of PEGylated recombinant methionyl human granulocyte colony-stimulating factor formulation approved for the treatment of severe chemotherapy-induced neutropenia [21].

All of the aforementioned formulations that have approved by the US FDA are classified as passive targeting nanotechnological platforms. Of the formulations categorized as active targeting platforms, to date only one has been approved by the US FDA [21]. The albumin-bound paclitaxel nanomedicine, Abraxane®, was approved as an injectable treatment of metastatic breast cancer [27, 53].

1.4 Current Uses in Cancer Diagnostic

Nanoparticle imaging agents can be used for noninvasive cellular and molecular imaging used to visualize targeted macromolecules or cells in vivo, particularly for magnetic resonance imaging (MRI), computed X-ray tomography (CT), optical imaging, positron emission tomography (PET), single-photon-emission computed tomography (SPECT), and ultrasound [54].

Generally, nanoparticle-based MRI contrast agents are comprised of three parts, the core nanoparticles which generate the signal enhancement, the water-dispersible shells which facilitate hydrophobicity and compatibility in vivo and the bioactive targeting ligands [54]. Targeting ligands, conjugated to magnetic resonance (MR) contrast probes, selectively bind to disease-specific biomarkers and facilitate the induction of a signal that provides different intensities between diseased and noncancerous cells [55].

Nanoparticle imaging agents offer enhanced contrast and favorable biodistribution. For example, superparamagnetic iron oxide nanoparticles are used as MRI contrast agents [24]. However, superparamagnetic iron platinum particles (SIPPs) have been reported and had significantly better T2 relaxivities compared with the more common iron oxide nanoparticles.

Methods used to fabricate nanoparticles for medical imaging are microemulsions, sol–gel syntheses, sonochemical reactions, hydrothermal reactions, hydrolysis and thermolysis of precursors, flow injection syntheses, and electrospray syntheses [56].

Furthermore, optical imaging windows are used to facilitate noninvasive intravital imaging of drug response in animal tissue [57]. They enable nonobtrusive intravital imaging in live tissue in so-called skin-fold chambers (SFCs), cranial imaging windows (CIWs), mammary imaging windows (MIWs), and abdominal imaging windows (AIWs). Repeated imaging is done through imaging windows, and it affords a noninvasive method to monitoring tumor behavior during the early, middle, and late progression of the disease stages or at various times following drug re-administration [57].

1.4.1 The Phosphatidylinositol 3-Kinase-AKT Pathway

Phosphatidylinositol 3-kinase (PI3 kinase) pathway is one of the important signaling pathways which play key roles in essential cellular processes including survival, proliferation, migration, and metabolism [58–60]. PI3 kinase pathway is a highly complex circuitry which regulates tightly the vital cellular mechanisms by controlling kinases, phosphatases and transcription factors. The kinases are the members of a family of lipid kinases which phosphorylate the inositol ring of phosphatidylinositides from the 3′-hydroxy position to produce phosphatidylinositol-3,4,5-trisphosphate (PIP 3) [60, 61]. PIP 3 is a well-known second messenger which accumulates AKT (protein kinase B) to the cell membrane [60, 61].

Not until mid-1980s, the significance of PI3 kinases was noticed in cancer studies [62]. Their transforming activity has resemblance with viral oncogenic tyrosine kinases as a result of shared structural and functional properties [62]. Following studies revealed that PI3Ks functions as heterodimer complexes which have both catalytic and regulatory subunits [63]. The catalytic subunits (p110) functions as kinases which phosphorylate the target molecules [64]. The regulatory subunit (p85), partnering with p110, is a target itself to a variety of cytoplasmic and receptor tyrosine kinases (RTKs). The interaction of p85 with the tyrosine kinases which target p85 has different mechanisms [61, 65]. They can associate through the consensus motif YXXM residues on the SRC homology 2 (SH2) domains of kinases [66]. Alternatively, the interaction can occur with the help of intermediate proteins as in the cases of the insulin receptor substrates (IRS1 and IRS2) [66].

There are eight kinase subunits detected in mammalians so far and they are grouped into three classes which have different isoforms, regulation mechanisms, structure, targets, and G-protein-coupled receptors [63]. Class I PI3 kinases phosphorylates phosphatidylinositol [61, 62] P 2 (also known as PIP 2) from the 3’ position to produce PIP 3 [61, 62]. Phosphatidylinositol’s (PtdIns) are the lipids that contain inositol. The class I PI3 kinases further divided into two groups, class IA (p110α, p110β, and p110δ) and class IB (p110γ). While group IA is triggered by mostly tyrosine kinases, group IB is regulated by G-protein-coupled receptors [63]. Studies showed that particularly class IA kinases play critical roles in cell growth and survival which also make them important targets for cancer initiation and progression. It is predicted that the gene that encodes p110α subunit is the most deregulated kinase in human cancers [70, 71]. The other classes of PI3 kinase family and their downstream targets including mTOR, DNA-PK, ATM, and ATR have crucial roles in DNA repair [61, 69, 70].

There are also three different genes that codes regulatory subunits with alternative isoforms that participate in class IA PI3 kinases (α, β, and γ) [64]. This subunit consists of an inter-SH2 domain between two SH2 domains, SH3 domain and a breakpoint cluster region (BCR)-homology domain at amino terminal [71]. Two subunits bind constitutively through inter-SH2 domain of p85. The alternative isoforms from regulatory subunit are p85, p55, and p50. The latter two isoforms do not have SH3 and BCR homology domains and they are considerably more active than p85 isoform due to the lack of inhibitory effect of SH3 domain [63].

PI3 kinase pathway is an active player of important cellular processes and their activity are tightly regulated. The pathway begins when PIP 2 is phosphorylated from 3′-hydroxy position by PI3 kinases to produce PIP 3 resulting in activation of the pathway [60, 61]. It is critical for cells to maintain PIP 3 levels at minimal rates when the signaling cascade is no longer needed. PTEN, which was previously known to involve in breast cancer and glioblastomas, is shown to be the primary phosphatase that converts PIP 3 back to PIP 2 by removing the phosphate group at the 3′-hydroxy position of PIP 3 [63]. Today, PTEN is a well-recognized TSG (tumor suppressor gene), and PTEN deactivation has been shown in many types of cancer. PTEN is the second most mutated TSG after p53 [70, 71].

PI3 kinases are produced and kept in normal cells in inactive forms form and their kinase activity is tightly regulated. The signaling cascade begins upon activation of RTKs by growth stimulation. RTKs recruit PI3 kinase heterodimer by binding to p85 regulatory subunit of PI3 kinase leading the p110 catalytic domain to convert PIP 2 to PIP 3. PIP 3 is a well-known lipid second messenger which transmits the signals by binding through pleckstrin homology (PH) domains of serine/threonine kinases AKT. At this step AKT is translocated into cell membrane with carboxyl-terminal modulator protein (CTMP) at its C terminal which prevents AKT from being phosphorylated [63]. CTMP is phosphorylated by a yet to be defined kinase to disassociate from AKT. In order to be fully active, AKT is then phosphorylated at Thr308 and Ser473 positions by PDK1 and PDK2, respectively [72, 73]. AKT activation initiates several mechanisms all of which have accumulative effects in cancer development. AKT prevents apoptosis by phosphorylating BAD, Caspase-9, and FKR [74, 75]. Cell proliferation is also promoted as a result of AKT activation because not only it works synergistically with RAS–MAPK pathway but also it prevents degradation of Cyclin D1 by phosphorylating glycogen synthase kinase-3β (GSK3β) [63]. Cell growth, which refers to increased cell mass or size by accumulated macromolecule synthesis, also is positively affected after AKT activation with unclear mechanisms involving mTOR [63, 76].

Considering the complexity and diversity of the PI3 kinase pathway members, it is not surprising to observe deregulation of PI3 kinase pathway in numerous cancer types. There are several modes of deregulation for different members in different cancer types [77–80]. It can be just overexpression of wild-type genes as in the case of ovarian cancer where p110 catalytic subunit of PI3K is overexpressed or AKT2 overexpression in breast, ovarian and pancreatic cancers. Truncated forms of p85 subunit which lack inhibitory domains may also lead to constitutively active PI3 kinase [67, 68, 81–83]. This type of deregulation is observed in colon and ovarian cancers. This pathway may also deregulate due to constitutively active stimulation from upstream signals. PTEN activity loss has been observed in numerous cancer types which show the critical position of PTEN in cell cycle regulations [71,72].

Over the years, the PI3 kinase pathway has been a target for specific chemical inhibitors [84]. These inhibitors gave promising results in in vitro studies; however, they usually fail in later stages. The reason for the failure is not clear