DRUG DELIVERY: FUNDAMENTALS AND APPLICATIONS

The field of theranostics (a portmanteau of “therapeutics” and “diagnostics”) aims to integrate therapeutics with diagnosis, in order to develop more individualized therapies. Life-threatening diseases, particularly high-risk cancers, are highly heterogeneous, so that treatments are typically effective for only limited patient populations and at certain stages of disease development. Merging of the paradigms of diagnosis and therapy will provide timely assessment of therapeutic response, allowing optimization of treatments and tailoring personalized medicine based on individual needs, to improve therapeutic outcomes. A current clinical example is the use of Herceptin^® (a monoclonal antibody used to treat patients with breast cancer), which is used in conjunction with the diagnostic tool, HercepTest^®. Herceptin^® targets the HER2 protein, which is overexpressed in approximately one-third of breast cancers. HercepTest^™ specifically demonstrates overexpression of the HER2 protein in breast cancer tissues and so is used to identify those patients who are most likely to benefit from Herceptin^® treatment.

Currently, research activities on theranostics are predominantly focused on cancer diagnosis and treatment. Cancer is the third most common cause of death in the world, following cardiovascular and infectious diseases. There are significant challenges in the successful delivery of cytotoxic drugs specifically to tumor cells while avoiding normal healthy tissues and minimizing side effects. In addition, it is essential to carry out diagnostic imaging to understand the cellular phenotypes, biological activity, and heterogeneity of each tumor. In response to these challenges, theranostics offers the potential to allow physicians to monitor the drugs given to each patient while assessing drug pharmacokinetics and biodistribution, as well as tumor response, all in a noninvasive and real-time manner. As a result, physicians will be able to avoid the prolonged use of nonresponsive therapies to treat cancer; instead, they will be able to alter or design innovative treatment regimens, tailored to each individual case, to improve overall survival.

FIGURE 18.1 Diagram outlining the basic principles of a theranostic agent. Theranostic agents possess both diagnostic and therapeutic elements in a single-nanoparticle construct.

Due to the continued advances in nanotechnology, including design and synthesis of new nanocarriers, the field of theranostics has evolved so that now it is directed more toward using multifunctional nanosystems, which simultaneously deliver both therapeutic and imaging moieties. Nanoparticles (NPs) can accommodate imaging probes, therapeutic agents, and/or targeting agents. This “all-in-one” approach allows assessment of both the pharmacokinetics and pharmacodynamics of the therapies. Figure 18.1 depicts the concept of theranostics based on NP platforms combining the therapeutic and diagnostic functions into one package.

The chapter will focus on the use of theranostics in cancer imaging and therapy. Medical imaging modalities are first introduced, followed by an overview of commonly used nanocarriers for drug delivery. A selection of multifunctional nanoplatforms for theranostic applications are then presented in detail.

18.2 MEDICAL IMAGING MODALITIES

Various types of medical imaging procedures allow the evaluation of organ functions and assist in the diagnosis of disease. Many of these imaging procedures are now being investigated for their application to theranostics.

Magnetic resonance imaging (MRI) produces images by measuring the radio-frequency signals that are given off by magnetized protons in living tissue. Image contrast of different tissues is created because protons in each tissue possess their own characteristic magnetic properties. Image contrast also can be enhanced with the aid of contrast agents that are delivered into the tumor tissue. Contrast agents are paramagnetic materials that can alter the magnetic properties of water protons to enhance the signal in the tissue of interest and help delineate it from that of the normal surrounding tissue. MRI is a beneficial imaging technique because it offers high spatial resolution without any tissue-penetrating limitations. Currently, highly stable gadolinium (Gd) complexes are commonly used to produce bright signal enhancement, while paramagnetic iron oxide nanoparticles (IONPs) generate dark signal enhancement.

Computed tomography (CT) is one of the most commonly used modalities in diagnostic imaging–based x-ray attenuation. CT provides superior visualization of structures with high densities but is limited when used to distinguish soft tissues that have similar densities. CT contrast agents are introduced in order to improve image contrast of soft tissue structures with similar or identical densities. CT contrast agents are composed of biocompatible materials containing elements of high atomic numbers. Clinical CT contrast agents are generally iodinated compounds. NPs containing high-atomic-number elements, such as gold, are now being investigated as CT contrast agents.

Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are nuclear imaging techniques used to map physiological and biological processes in the body following the administration of radiolabeled tracers. In PET, positrons (positively charged electrons) emitted from the nucleus during decay travel a few millimeters in the tissue, where they undergo annihilation by colliding with electrons. Each annihilation event releases two gamma-ray photons of equal energy (511 keV) in opposite trajectories (180° apart). PET scanners utilize the simultaneous detection of these two photons to precisely locate the source of the annihilation event. Positronemitting isotopes ¹⁵O, ¹³N, ¹¹C, ¹⁸F, ⁶⁴Cu, ⁶²Cu, and ⁶⁸Ga are often incorporated into biorelevant materials to follow their distribution and concentration, and to detect malignant diseases. SPECT employs gamma-emitting isotopes, such as ^99mTc, ¹¹¹In, and ¹²³I, as probes to detect biomarkers and to label NPs. SPECT imaging can quantitatively determine disease-related biomarkers for diagnostic imaging, to track biodistribution of NPs and to assess therapeutic efficacy.

Optical imaging utilizes photons of a characteristic wavelength to excite fluorescence materials, to visualize biomarkers and to study the localization of materials. Optical imaging offers a high sensitivity and the capability of multicolor imaging. A major limitation of optical imaging is the small depth of light penetration, ranging from hundreds of microns to several centimeters. Near-infrared (NIR) imaging has proven to be more desirable owing to better tissue penetration and less interference from tissue autofluorescence.

18.3 NANOCARRIERS FOR THERANOSTICS

Nanocarriers for theranostics may be constructed from a wide range of organic and inorganic materials, including lipid-based systems (including liposomes, polymersomes, polymeric micelles), polymeric NPs, gold NPs, iron oxide NPs, quantum dots (QDs), silica NPs, calcium phosphate NPs, and various types of carbon nanomaterials (including carbon nanotubes [CNTs]). These carriers have been widely investigated as carriers for drug targeting and delivery and, as such, are described in detail in Chapter 5. The drug carriers described in this chapter can be seen as an extension of this research, whereby the drug delivery system incorporates not only a drug but also imaging agents (contrast agents, fluorescent probes, and radiolabels), for use in diagnostic imaging. Some of these nanocarriers also are cytotoxic via magnetic hyperthermia and photothermal ablation, as well as photodynamic therapy (PDT).

A particular advantage that NPs offer for theranostic purposes is that they can be targeted to specific disease sites, e.g., to the tumor/tumor cells, thereby reducing unwanted side effects. Nanocarrier targeting is described extensively in Chapter 5; to summarize here, targeting can be achieved by either (1) passive or (2) active means. Passive targeting exploits the natural distribution profile of an NP in vivo. Due to their size, shape, charge, and other physicochemical characteristics, NPs tend to be recognized as “foreign” by the immune system and are taken up by the reticuloendothelial system (RES). Their accumulation in RES organs such as the liver and spleen is desirable if these are the target organs. Alternatively, NPs can be sterically stabilized, by rendering their outer surfaces more hydrophilic. Typically, this involves covalently attaching polyethylene glycol (PEG) chains to the NP surface. The highly hydrated PEG layer reduces opsonization and RES uptake, thereby prolonging NP circulation time in the blood. This “stealth” technology can then allow sufficient time for the nanocarriers to accumulate at a tumor site, via what is termed the “enhanced permeability and retention” (EPR) effect of tumor vasculature (Maeda et al. 1984). Tumor blood supply is associated with irregularly dilated and leaky blood vessels, with large pore sizes. NPs that are smaller than 100 nm (although it should be noted that the precise particle size is not fully clear and is a subject of debate—see, for example, Chapter 5, Section 5.2.1) can thus extravasate into the tumor tissue, by passing through the pores and preferentially accumulating there.

Active targeting is the use of a specific-targeting ligand, in order to improve uptake and sequestration into the tumor. The introduction of targeting ligands can help increase the target-to-background contrast in medical imaging while also improving the local concentration of the therapeutics at the tumor site and reducing its systemic toxicity. A targeting moiety is often covalently attached to the NP surface. Ideally, a ligand is used which targets a protein that is predominately overexpressed on tumor cells, in order to prevent the delivery vehicle from accumulating elsewhere in the body. For example, folic acid is often used as a targeting motif, as the folic acid receptor is known to be overexpressed on cancer cells.

Due to their very high surface-area-to-volume ratios, NPs have high loading capacities. Drugs and imaging agents can be loaded into nanocarriers. Encapsulated within the nanocarriers, these agents are protected from in vivo degradation, allowing controlled drug release. The NP surface can be modified with targeting ligands, drugs, imaging agents, stealth coatings, etc. Furthermore, the use of external stimuli, including temperature, light, and magnetism, can create activatable NPs, which allow for the selective trigger of drug release and image-based mechanisms. Some NPs demonstrate a pH-dependent solubility profile, which can also be used to control drug release.

18.4 INORGANIC-NANOSIZED THERANOSTICS

18.4.1 GOLD NANOPARTICLE–BASED THERANOSTICS

Gold nanoparticles (AuNPs) have emerged as one of the most extensively investigated theranostic platforms for the diagnosis, imaging, monitoring, and treatment of malignant and other diseases. AuNPs possess a variety of advantageous properties that make them very useful as multifunctional theranostic vehicles. AuNPs offer inherent biological compatibility, an important advantage over many other synthetic delivery systems described later. Furthermore, their large surface area means they can serve as highly efficient carriers, capable of high drug and diagnostic loading. Using simple wet-laboratory techniques, gold nanocarriers have been constructed in a variety of shapes and sizes, including spheres, cubes, rods, cages, and wires; all of which can now be prepared with accurate quality control and in large quantity. They can also be used as either the core or the shell, for polymer–metal and metal–metal hybrid NPs.

AuNPs have high atomic number and induce strong x-ray attenuation, making them effective contrast agents for CT imaging. Because AuNPs are visible by CT, they have potential to be noninvasively assessed in pharmacokinetics, biodistribution, and tumor targeting. AuNP-based theranostics are often developed by surface modification with therapeutic agents and other imaging agents. Photosensitizers (PS), dyes, drugs, and targeting ligands can all be attached to the surface of AuNPs either directly, via amine or thiol groups, or indirectly, using a linker molecule such as bovine serum albumin. Other imaging agents, such as Gd chelates for use in MRI, and radioisotopes for nuclear medicine, are attached to AuNPs for multimodal imaging and image-guided therapy.

Encouraging preclinical data is accumulating on the use of AuNPs as theranostic agents. Chen et al. have recently synthesized and characterized folic acid–functionalized, dendrimer-entrapped AuNPs containing the MRI agent Gd, thereby developing a nanoprobe suitable for both CT and MRI methodologies (Chen et al. 2013). The AuNPs were entrapped within the dendrimer interior and the Gd was modified on the dendrimer surfaces. This multifunctional construct specifically targeted folic acid receptor–expressing cancer cells via a receptor-mediated pathway. Both in vitro cell imaging and in vivo tumor imaging demonstrated significantly improved contrast-to-noise ratios, indicating the potential of this system to detect cancer cells in the body using dual-mode CT/MRI.

AuNPs can undergo surface plasmon resonance. This is an optical phenomenon that arises from the interaction between an electromagnetic wave and the conduction of electrons in a metal. When excited with light at, or near, the absorption maximum (typically in the visible, or NIR, range), the electrons in AuNPs collectively absorb the incoming irradiation and are excited from the ground state to a higher energy level, where they oscillate at a particular resonance frequency. The nonradiative energy relaxation of the electrons that back down to their ground state results in an increase in kinetic energy and ultimately leads to the generation of intense heat into the local environment. Thus, AuNPs can act as energy transducers, converting the absorbed light into heat (see also Chapter 14, Figure 14.18). This photothermal effect can be harnessed for cancer therapy: AuNPs accumulate at a tumor site and are subsequently illuminated; the heat spike can then selectively damage the tumor tissue, while normal tissue is unharmed. In this application, the AuNPs are not functioning as carriers for a cytotoxic drug; rather the AuNPs themselves are causing tumor cell death, via hyperthermia.

Spherical AuNPs, with a characteristic absorption at 500–600 nm, are not appropriate materials for such an application. In contrast, if the morphology is changed to a hollow AuNP construct, such as a nanocage or nanoshell, this can shift the absorption to the NIR region, between 600 and 1000 nm. This is the therapeutic window whereby the interaction of light with biological tissues is low, keeping attenuation and scattering effects to a minimum, reducing unwanted interactions with the surrounding healthy tissue.

Reports on the success of hollow gold nanospheres (HAuNS) for photothermal ablation are increasing. For example, Melancon et al. conjugated the targeting ligand C225 (an antibody directed at epidermal growth factor receptor) to the surface of HAuNS and then evaluated their distribution in mice (Melancon et al. 2008). It was shown that C225-HAuNS had a significantly higher uptake in the tumors, compared with IgG-conjugated HAuNS controls. The efficacy of photothermal ablation was also assessed. Magnetic resonance thermal imaging revealed that for mice injected with C225-HAuNS, the exposure to low doses of NIR light resulted in average maximum temperatures of 65°C, whereas the saline control mice resulted in an average maximum temperature of only 47° after laser treatment. Furthermore, histological analysis showed that tumors treated with the C225-coated HAuNS developed significantly larger necrotic areas than the control tumors following exposure to the NIR laser.

Gold nanorods (GNRs) have also demonstrated potential for photothermal ablation, due to their high light absorption coefficient in the NIR region, good photothermal stability during laser illumination, and high heat generation. GNRs bearing a folate ligand for cancer cell targeting were also labeled with radioactive iodine, in order to monitor NP distribution in vivo during the treatment period (Jang et al. 2012).

18.4.2 IRON OXIDE NANOPARTICLE–BASED THERANOSTICS

IONPs are made from magnetite (Fe₃O₄). IONPs less than 20 nm are superparamagnetic (i.e., the particles show zero magnetism in the absence of an external magnetic field but can become magnetized in the presence of one) and can be used as contrast agents in MRI. Unlike Gd-based contrast agents, superparamagnetic iron oxide nanoparticles (SPIONs) decrease MR signals of surrounding water protons, resulting in dark signal enhancement. SPIONs are extensively investigated for theranostic purposes. SPIONs need to be surface-engineered to improve their solubility, stability, and performance in vivo. SPIONs are typically coated with hydrophilic materials. A wide variety of coating materials have been used for SPION modification, including dextrans, dendrimers, and polyvinylpyrrolidone. Therapeutics and targeting agents have been covalently conjugated to SPIONs. Biodegradable spacers, e.g., peptides, have been used to facilitate drug release in response to the environment, e.g., within lysosomes. This ensures release of the drug into the cytosol of cells, upon reaching the acidic endosome/lysosome compartments after cellular uptake (Kohler et al. 2005). Small-sized SPIONs are often incorporated into other larger drug carriers, including liposomes, polymersome, polymeric NPs, and silica NPs, to enable visualization of the larger carriers noninvasively with MRI.

Similar to the photothermal ablation described in Section 18.4.1 for AuNPs, magnetic NPs also can be used as therapeutic entities per se, rather than serving as carriers for a therapeutic drug. In this case, the cytotoxic effect is due to magnetic hyperthermia, which uses a combination of both alternating magnetic fields and magnetic NPs as heating agents, to induce a localized and specific heat around a tumor region. SPIONs possess significant energy absorption properties due to the Neel relaxation phenomenon. When placed in alternating magnetic fields, SPION dipole moments are quickly reoriented, depending on the frequency, magnetic field strength, NP size, and environmental temperature. The electromagnetic energy is dissipated as heat, and the resulting temperature spike within the surrounding tissue can be exploited for hyperthermia-induced destruction of cancer cells.

SPION clusters have been prepared that are surface derivatized with folic acid for tumor cell targeting and also contain an outer PEG shield, to promote long circulation times and tumor accumulation. After i.v. injection, the SPION clusters accumulated locally in cancer tissues within the tumor and enhanced the MRI contrast (Hayashi et al. 2013). On the application of an AC magnetic field, it was found that the temperature of the tumor was approximately 6°C higher than the surrounding tissues, 20 minutes after treatment. Thirty-five days after treatment, the tumor volume of treated mice was one-tenth that of the control mice. Furthermore, the treated mice were alive after 12 weeks, whereas control mice died up to 8 weeks after treatment.

Localized magnetic hyperthermia can also be used to trigger controlled drug release from a nanocarrier. Three functionally different polymers were used to surround SPIONs and generate a thermosensitive nanocarrier (Rastogi et al. 2011): (1) the temperature-sensitive polymer, N-isopropylacrylamide; (2) poly(acrylic acid), to tune the critical temperature point and enhance conjugation to the NP surface; and (3) PEG-methacrylate, to provide a stealth coating to the NP, increasing the circulation time and introducing reactive hydroxyl groups for the coupling of folic acid. The folic acid was incorporated as a targeting ligand for cancer cells. The polymeric nanostructures, loaded with doxorubicin (Dox), were approximately 200 nm in size and functioned as MRI contrast agents in phantom gels. The thermo-responsive property of the carrier resulted in controlled release of Dox following SPION-induced hyperthermia, contributing to a release rate that was nearly 2.5-fold higher than that for normal physiological conditions, during the first 48 hours.

18.4.3 QUANTUM DOTS THERANOSTICS

QDs are luminescent semiconductor nanocrystals, typically with cadmium–selenium (CdSe) or cadmium–tellurium cores. For imaging purposes, QDs are proving particularly valuable for multicolor fluorescent applications. Some of the major advantages of using QDs over traditional fluorescent dyes include their high absorbance, narrow and symmetric emission bands, stability, and resistance to photobleaching (Resch-Genger et al. 2008). It is also possible to control their optical properties by simply tuning the particle size (the larger the dot, the redder its fluorescence spectrum). In addition, QDs are able to form fluorescence resonance energy transfer (FRET) pairs with fluorescent dyes, allowing their use for monitoring the intracellular trafficking of particles.

As well as their use as imaging agents, the high surface-area-to-volume ratio of QDs enables the construction of a multifunctional nanoplatform, where the QDs serve not only as an imaging agent but also as a nanoscaffold for both therapeutic and diagnostic modalities. To this end, there is now a considerable body of literature describing how QDs have been functionalized with a variety of drugs, including cytotoxics, as well as targeting moieties, including aptamers, antibodies, oligonucleotides, peptides, and folates (Ho and Leong 2010).

A promising example of the potential of QDs as theranostic carries is the development of a QD–RNA aptamer–doxorubicin conjugate (QD–Apt(Dox)) for synchronous cancer imaging and traceable drug delivery (Bagalkot et al. 2007). In this system, the RNA aptamer serves as a targeting ligand for the prostate-specific membrane antigen, expressed in LNCaP cells. Dox was intercalated into the aptamer, resulting in a multifunctional QD platform for imaging and therapy. QD–Apt(Dox) has demonstrated enhanced therapeutic specificity in vitro against LNCaP cells, compared to nonspecific PC3 cells.

One elegant approach has been the in situ immobilization of CdSe QDs in the interior of a pH-and temperature-dual responsive hydroxypropylcellulose–poly(acrylic acid) (HPC-PAA) nanogel (Wu et al. 2010). In this multifunctional platform, the fluorescent QDs act as an optical identification code for sensing and imaging (Figure 18.2). The hydroxypropylcellulose (HPC) chains provide rich –OH groups for sequestering Cd²+ ions into the gel network, thereby stabilizing the QDs embedded in the gel network and minimizing their toxicity. The presence of the hydrophilic HPC chains on the surface of QDs also provides steric stabilization, to reduce opsonization and subsequent uptake by the RES system in vivo. High drug-loading capacity of the anticancer drug temozolomide (TMZ) was attributed to hydrogen bond interactions between TMZ and (1) the carboxyl groups in polyacrylamide (PAA) network chains and (2) the hydroxyl groups in HPC chains. The pH-sensitive PAA network chains were designed to induce a pH-responsive volume phase transition of the nanogel, in order to facilitate stimulus-responsive drug release. At higher pH levels, the carboxylic acid groups of the nanogels become deprotonated, which enhances nanogel swelling, thereby facilitating pH-sensitive drug release. Higher pH levels also destroy PAA–drug hydrogen bonds, further promoting drug release from the system. In vitro cytotoxicity tests indicate that the empty hybrid nanogels have very low cytotoxicity, whereas the TMZ-loaded hybrid nanogels have high anticancer activity.

FIGURE 18.2 Schematic representation of HPC–polyacrylamide–quantum dot hybrid nanogels, for multifunctional application in drug delivery and fluorescence quantum dot imaging (HPC, hydroxypropylcellulose; PAA, polyacrylamide; CdTe, cadmium-tellurium). (Reprinted from Biomaterials, 31(11), Wu, W., Aiello, M., Zhou, T. et al., In-situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery, 3023–3031, Copyright 2010, with permission from Elsevier.)

Despite their impressive potential as future theranostic agents, a major disadvantage of the use of QDs in biomedical applications is their inherent cytotoxicity. Surface oxidation, and the leaching out of heavy metal ions from the core, are serious concerns. However, new strategies to minimize cytotoxicity are being developed. QDs can be associated with other theranostic carriers, to form many other types of multifunctional platforms for imaging and therapy. The association of QDs with liposomes, micelles, and CNTs is described later in this chapter. The combination of the QD with another carrier can significantly reduce the cytotoxicity of the QDs in vivo.

18.4.4 MESOPOROUS SILICA NANOPARTICLES

A mesoporous material is one that contains pores with diameters between 2 and 50 nm. Mesoporous silica nanoparticles (SiNPs) are typically of size 100–200 nm and are showing considerable promise as theranostic carriers. Silica is generally regarded as a biosafe material and is used clinically in implants. The ultrahigh surface area of mesoporous SiNPs allows for extensive surface modification with targeting ligands and solubilizers; their porous interior enables them to serve as ample reservoirs, both for therapeutic drugs and imaging agents. Surface attachment with hydrophilic groups increases the solubility and also the stability of the NP dispersion in aqueous solutions.

Mesoporous SiNPs, of size 100–200 nm, were doped with SPIONs for magnetic manipulation and MRI, as well as the hydrophobic anticancer drugs camptothecin and paclitaxel (Liong et al. 2008). The surfaces of these particles were functionalized with hydrophilic phosphonate groups, in order to achieve high stability and solubility in aqueous environments. Additionally, the fluorescent dye, fluorescein isothiocyanate, was conjugated onto the surface for optical imaging capabilities, and folic acid groups were attached to the surface to promote uptake by tumor cells. Cellular uptake studies in two types of pancreatic cancer cell lines indicated that the NPs were internalized within 30 minutes of transfection and exhibited greater toxicity levels than drug-free SiNP controls.

18.4.5 CALCIUM PHOSPHATE NANOPARTICLES

Calcium phosphate nanoparticles (CPNPs) offer a number of advantages as theranostic nanocarriers. A particular advantage over many other synthetic theranostic systems is their nontoxicity: both Ca²⁺ and PO₄³⁻ are found in relatively high concentrations in the body. The formation of CPNPs is a relatively straightforward precipitation reaction, so that encapsulation of the cargo molecules merely necessitates that the molecule of interest is present during NP formation. Additionally, CP is an easily substituted matrix, permitting the inclusion of a broad variety of substitutions, such as organic fluorophores. Fluorescent dyes can be used as a tracking device to follow the fate of the NPs in vivo and give an observable indication of cargo delivery. Encapsulation in the CP matrix can protect the drug cargo in vivo, and the incorporation of tumor-specific targeting ligands can facilitate uptake by cancer cells.

Furthermore, calcium phosphates, regardless of calcium/phosphate ratio, crystallinity, or phase, demonstrate a pH-dependent solubility, being relatively insoluble at physiological pH 7.4, but becoming increasingly soluble below pH 6.5. This pH-tunable solubility has been a major impetus in the development of CPNPs for targeting and controlled-release purposes. CPNPs can dissolve to release their cargo in the acidic environment that commonly prevails in the vicinity of solid tumors, thereby facilitating drug release in the vicinity of the tumor. CPNPs that have been taken into cells and become localized within the acidic endosomal compartment will also dissolve, releasing drug intracellularly. Thus, drugs normally with little or no solubility in physiological liquids can be delivered intracellularly, using CPNPs.

A double reverse-micelle strategy has been developed to prepare stable, nonaggregating, 20 nm CPNPs embedded with fluoroprobes and the small amphiphilic neoplastic drug, ceramide, for simultaneous bioimaging and drug delivery to a range of cell types, including melanoma and breast adenocarcinoma cell lines (Kester et al. 2008). The lifetimes and quantum properties of the fluorescent dye were shown to improve when encapsulated within the CPNPs. Furthermore, ceramide encapsulation was able to induce apoptosis, as measured by an MTS assay, in vitro. In further studies, the CPNPs were functionalized with PEG groups and targeting antibodies (Barth et al. 2011). In vivo imaging using NIR microscopy of the encapsulated indocyanine green fluorophore indicated greater tumor accumulation of targeted NPs than their nontargeted counterparts, as well as significantly better penetration through the blood–brain barrier.

18.4.6 CARBON NANOMATERIALS

Based on their structures, carbon nanomaterials are classified into a variety of categories that include fullerenes, carbon dots, nanodiamonds, carbon nanotubes (CNTs), and graphene. All of these systems have been investigated for applications in theranostics. CNTs are extensively investigated in theranostics and discussed here as an example. Structurally, single-walled carbon nanotubes (SWCNTs) are a single sheet of graphene, rolled seamlessly into a cylinder, while multiwalled carbon nanotubes (MWCNTs) comprise concentrically layered SWCNTs of increasing diameter. CNTs possess a high aspect ratio, with diameters typically in the nanometer range (0.5–3 nm [SWCNTs] and 2–100 nm [MWCNTs]), but lengths that can extend to several micrometers.

The graphite-like structure of CNTs is inert and thus not suitable for most conjugation chemistry. Researchers have applied extreme oxidative conditions to functionalize the surface, which can subsequently be utilized as mounting sites. Various molecules, including peptides and antibiotics, have been conjugated to the CNT surface in this way. CNT surfaces are also modified by both covalent (chemical conjugation of hydrophilic polymers) and noncovalent (via the physical adsorption of surfactants, such as sodium dodecylbenzene sulfonate) means, in order to improve their solubility and biocompatibility.

FIGURE 18.3 Schematic of a carbon nanotube loaded with the anticancer drug paclitaxel and functionalized with a poly(lactide-co-glycolide) (PLGA) polymer coating and luminescent quantum dots. (From Guo, Y., Shi, D., Cho, H. et al.: In vivo imaging and drug storage by quantum-dot-conjugated carbon nanotubes. Adv. Funct. Mater. 2008. 18. 2489–2497. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.)

CNTs containing an ultrathin poly(lactide-co-glycolide) (PLGA) coating were prepared by a novel plasma polymerization method (Guo et al. 2008). The PLGA coating facilitated the conjugation of amine-containing QDs onto the surface of the CNTs, for in vivo imaging. The CNT-QD nanoconstructs were loaded with the anticancer drug paclitaxel (Figure 18.3) and demonstrated in vitro antitumor efficacy against human PC-3MM2 prostate cancer cells. They were also successfully injected in mice for in vivo optical imaging. Studies indicated predominant CNT-QD uptake in the liver, kidney, stomach, and intestine.

CNTs also demonstrate strong optical absorbance in the NIR region. The emitted photoluminescence can be used for in vivo tumor imaging while also acting as an efficient NIR absorber and heater for photothermal ablation of tumors with a low injection dose. SWNTs are able to increase the local temperature of a tumor to as high as 60°C within less than 5 minutes of 808 nm laser irradiation. Compared to other thermal ablation platforms, such as AuNPs, SWNTs can effectively eliminate tumors at 10-fold lower doses and at lower irradiation powers (Moon et al. 2009).

The primary hindrance to employing CNTs for in vivo biomedical applications arises from potential toxicity issues, which include oxidative stress and inflammatory pathways (Rothen-Rutishauser 2010). A considerable research effort is being directed toward methods to reduce toxicity, including the use of polymeric coatings on the CNT surface.

18.5 ORGANIC-NANOSIZED THERANOSTICS

18.5.1 LIPOSOMES

Liposomes (described extensively in Chapter 5, Section 5.5.1) consist of closed, spherical vesicles, composed of one or more lipid bilayers, surrounding an aqueous core (Figure 18.4a; see also Figure 5.9). With a typical size range of approximately 80–200 nm, they provide ample cargo room for the incorporation of both drug molecules and diagnostic agents. Hydrophilic molecules can be accommodated within the aqueous liposomal core, while hydrophobic species can be associated with the lipid bilayers. Liposomes are widely used and highly successful drug carriers; commercially available liposomal drug delivery systems include AmBisome^®, DaunoXome^®, and Doxil^®. Liposomes can also be loaded with a variety of imaging agents for MR, nuclear, and fluorescence imaging applications.

FIGURE 18.4 Therapeutic molecules and imaging agents can be simultaneously encapsulated into (a) liposomes or (b) polymeric nanoparticles. Depending on their amphiphilicity, the drug and imaging agents can be encapsulated within the aqueous core or the hydrophobic bilayer of liposomes. The particle surface of both lipid and polymeric particles can be conjugated with ligands, for targeted delivery, and with PEG, for increased circulation time in the bloodstream (PEG, polyethylene glycol).

Liposomes offer the ability to protect the encapsulated agents from harsh external environments, to prolong their systemic circulation lifetime, and to be functionalized with various targeting ligands for cell- or tissue-specific delivery. Long-circulating “stealth” constructs can be prepared by covalently attaching PEG chains to the liposomal surface.

Although still at an early stage of development, some examples serve to demonstrate the potential of liposomes as theranostic agents. A PEGylated cholesterol/DOPE/DSPC liposome was used as a theranostic carrier for Dox and IONPs as MRI contrast agents (Erten et al. 2010). In this formulation, Dox was loaded into a dextran hydrogel placed between the iron oxide core and the lipid shell. The liposomes were studied in mice bearing lung and pancreatic carcinoma xenografts and demonstrated enhanced MRI contrast. Furthermore, the IONPs also served as a heating source upon the exposure of alternative magnetic field, providing a platform for localized hyperthermia-induced drug release, in addition to their role as an MRI contrast agent (Tai et al. 2009).

Liposome-based theranostic carriers can also be used for radionuclide imaging. A PEGylated liposome formulation was loaded with a chemotherapeutic agent and a copper radionuclide (⁶⁴Cu) suitable for PET imaging (Petersen et al. 2011). The pharmacokinetics of this agent was studied in flank HT29 colon tumors. The accumulation of ⁶⁴Cu liposomes in the tumors was several orders of magnitude higher than that of the radionuclide bound to a chelating agent (⁶⁴Cu-DOTA). In addition, the ⁶⁴Cu liposomes were able to remain in the blood for over a 24-hour period of time, indicating the long-circulating nature of the PEGylated NPs, in comparison to their nonliposomal counterparts.

QDs have been associated with liposomal carriers, for use in fluorescence imaging. The fabrication and characterization of a liposomally encapsulated QD-FRET probe for monitoring the enzymatic activity of phospholipase A2 has been described (Kethineedi et al. 2013). To fabricate the probes, luminescent QDs capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes (average diameter approximately 100 nm). Incorporating TOPO-capped QDs in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The probes were able to detect very low amounts of phospholipase A2 and to monitor enzyme activity in real time.

18.5.2 POLYMERSOMES

Liposomes are metastable assemblies, in which the lipid molecules can migrate between the inner and outer layers of the lipid membrane. This may result in leakiness of their core contents, in addition to liposomal fusion and fission, which are all sources of liposomal polydispersity and instability. Polymeric liposomes, or polymersomes, are a more recent variation of liposome carriers, which use amphiphilic synthetic block copolymers to form the vesicle membrane. The polymer chains in polymersomes can be tailored to enable cross-linking within the construct, in order to eliminate the “flip-flop” migration between bilayers that can occur in liposomes. Desirable functional groups can also be incorporated into the polymer chains, for the subsequent conjugation of drug molecules and imaging agents.

Spherical and wormlike polymersomes have been studied for the combined delivery of an anticancer drug and an MRI contrast agent (Yang et al. 2010a,b). Heterobifunctional asymmetric triblock copolymers were synthesized, comprising folate-PEG5000-b-poly(glutamate hydrazone doxorubicin)-b-PEG2000-acrylate and folic acid-PEG114-b-PLA-b-PEG46-acrylate. These formed a self-assembling vesicular polymersome structure, with inner and outer hydrophilic PEG layers. The long PEG segments were mostly segregated in the outer hydrophilic leaflet of the membrane, whereas the shorter PEG block was mostly present in the inner hydrophilic layer. The polyglutamate segment formed the hydrophobic membrane of the vesicles, to which the chemotherapeutic agent Dox was conjugated, via a pH-sensitive hydrazone bond, in order to achieve pH-responsive drug release. Targeting was achieved by conjugating folic acid ligands onto the outer PEG segment. The polymersomes were stabilized by cross-linking the polymeric methacrylate block in the interior of the membrane bilayer. Hydrophilic SPIONs were encapsulated into the aqueous core, to allow for MRI detection. The polymersomes showed higher cellular uptake by HeLa cells than folate-free polymersomes, due to the folate receptor–mediated endocytosis process. Accordingly, Dox-loaded polymersomes exhibited higher cytotoxicity than the folate-free controls and higher contrast for MRI.

18.5.3 POLYMERIC NANOPARTICLES

A wide range of polymeric NPs have been investigated as theranostic carriers (Figure 18.4b). Polymeric NPs have been described extensively in Chapter 5 (Section 5.5.5). Many different polymers can be used in the preparation of NPs. Both the polymer cores and the NP surface can be loaded with a variety of therapeutic or imaging agents. To ensure stability while minimizing immu-nogenicity, polymeric NPs can be shielded by stealth materials, much like their lipid counterparts. Targeting moieties can be conjugated to the constructs, to enhance tumor uptake. Sustained and controlled release of these agents can be achieved by surface or bulk erosion, diffusion through the polymer matrix, swelling followed by diffusion, or stimulation by the local environment.

Chitosan and cyclodextrin polymers are derived from natural resources and have received extensive attention due to their biocompatibility. Theranostic chitosan NPs have been prepared, carrying both the NIR dye Cy5.5 for live imaging, and the hydrophobic cytotoxic drug paclitaxel, for cancer treatment (Na et al. 2011). The hydrophobicity of water-soluble glycol chitosan was increased by chemically modifying the polymer with 5β-cholanic acid so that the polymer would form NPs in the water and encapsulate the hydrophobic drug.

A significant amount of research is also ongoing in the design of polymeric NPs using synthetic polymers that are biocompatible and biodegradable. The most studied polymers are those based on PLGA. Ling et al. (2011) prepared theranostic multifunctional PLGA NPs, loaded with Dox and IONPs for MRI contrast. Drug-release experiments demonstrated the efficacy of the PLGA NPs to release the drug in a controlled manner. PC3 cells treated with this system, bearing a targeting moiety, were characterized by higher intracellular iron concentration and stronger MRI contrast effects, in comparison to cells that were treated with nontargeted analogs.

18.5.4 MICELLES

Polymeric micelles are prepared using block copolymers and consist of an inner hydrophobic core and an outer hydrophilic, biocompatible, shell (see also Chapter 5, Section 5.5.6). Various types of hydrophobic therapeutic and imaging functionalities can be introduced into the core of the structure, while the hydrophilic shell confers biocompatibility, stability, and extended circulation times. Multifunctional polymeric micelles have been developed as theranostic agents, composed of PEG-poly(glutamic acid), which incorporate Gd-based MRI contrast agents and platinum anticancer drugs in their core. These micelles were found to localize in the interior of an orthotopic pancreatic lesion in a mouse model (Kaida et al. 2010).

A targeted polymeric micellar platform was developed, composed of amphiphilic maleimide- and methoxy-terminated PEG-poly(lactide) block copolymers (MAL-PEG-PLA and MPEG-PLA) as carriers for the cytotoxic Dox, and SPIONs as contrast agents for MRI. The arginine-glycine-aspartic acid (RGD) tripeptide, which targets an integrin receptor, was used as a targeting ligand. A thiol–maleimide reaction scheme was used to functionalize the micelles with the α_vβ₃ integrin receptor–targeting cRGD peptide. Different levels of cRGD loading were achieved by controlling the amount of MPEG-PLA introduced into the system. Conjugating the targeting peptide onto the particle was able to increase cellular uptake, MRI contrast, and Dox-induced cytotoxicity in vitro (Nasongkla et al. 2006).

QD-loaded micelles have been developed to increase the biocompatibility and stability of these optical agents. In one study, a hydrophobic lipid (10,12-pentacosadiynoic acid [PCDA]) was incorporated into the micellar structure in order to facilitate drug encapsulation (Nurunnabi et al. 2010). In addition, this lipid was able to create a strong outer shell, due to its polymerizable capabilities upon UV irradiation. Both PEG-PCDA and PEG-herceptin conjugates were incorporated into the final cross-linked micellar carrier. By enabling better uptake and retention in the cancerous lesion, the QD-loaded micelles exhibited enhanced tumor activity and selective toxicity, yielding a significant reduction in tumor volume.

18.6 MULTIFUNCTIONAL-NANOSIZED THERANOSTICS FOR PHOTODYNAMIC THERAPY

Various references have been made previously to the use of magnetic hyperthermia (via SPIONs) and photothermal ablation (via AuNPs and CNTs), as methods to induce localized and specific cell death. A further approach, known as photodynamic therapy, involves the delivery of a photosensitizer (PS) to tumor tissues, followed by their irradiation with a laser of appropriate wavelength. Upon irradiation, activated PS convert molecular oxygen to toxic singlet oxygen and free radicals (reactive oxygen species), which induce apoptosis, cell death, and tissue destruction. Nontarget toxicity can be prevented with this technique because the activation of cytotoxic species only occurs at the site of illumination, which allows the therapy to be localized. A number of different theranostic carriers have been developed utilizing this approach, in which multifunctional platforms are prepared, containing a PS (as the therapeutic agent), an imaging agent, and, optionally, other components, such as targeting ligands, and stealth coatings.

Porphyrin derivatives are the most commonly used PS in PDT. A PDT theranostic agent for the imaging and treatment of brain tumors has been developed, using the PS Photofrin^®, a complex mixture of porphyrin oligomers (Kopelman et al. 2005). The PS was incorporated into a PAA core, along with MRI contrast materials. The NP was further functionalized with PEG groups and RGD-targeting peptides. The major advantages of encapsulating Photofrin^® with the NPs are (1) protection of the PS from degradation in vivo, (2) reduced cutaneous photosensitivity posttreatment, and (3) the waiting time between i.v. injection of the PS and subsequent laser irradiation is greatly reduced. In vivo pharmacokinetic behavior, studied in a gliosarcoma rat model by diffusional MRI, revealed a 50-fold increase in NP circulation half-life after PEGylation of the NPs. In addition, a significant increase in the diffusion coefficient of the water surrounding the tumor cells was observed, indicating a decrease in tumor growth following treatment. The PDT-Photofrin-PAA NPs were able to effectively kill engrafted brain tumors in rats within 5 minutes of light exposure.

Multifunctional NPs have been described consisting of a gold–silver nanocage core, surrounded by a silica shell containing the NIR PS, Yb-2,4-dimethoxyhematoporphyrin (Yb-HP). This PS allowed monitoring of tumor growth, as well as simultaneously administering therapy by PDT and plasmonic heating (Khlebtsov et al. 2011). Significant death of HeLa cervical cancer cells occurred in vitro when they were incubated with the NPs and irradiated with light. This was due to both the plasmonic photothermal heating effects of the gold–silver nanocages, in addition to the photodynamic effects of the Yb-HP.

PEGylated poly-(L-glutamic acid) conjugates containing a PS, as well as a Gd-based MRI contrast agent, have been developed (Vaidya et al. 2008). The MRI contrast agent provided both image guidance for the precise application of laser irradiation at the target site and noninvasive assessment of the therapeutic efficacy of PDT. MRI images revealed that PEGylated constructs achieved longer blood circulation times, lower liver uptake, and greater tumor accumulation than their non-PEGylated counterparts. Furthermore, PDT-treated animals that had been administered the PEGylated conjugates showed greater tumor growth inhibition.

A targeted PTD agent with a built-in apoptosis sensor (TaBIAS) has been designed, which both triggers and images apoptosis in cancer cells (Stefflova et al. 2006). The nanostructure consists of four components (Figure 18.5): (1) a PS, pyropheophorbide a, which localizes near mitochondria; (2) a fluorescence quencher Black Hole Quencher-3, which quenches the PS’s fluorescence; (3) an enzyme-cleavable peptide linker (susceptible to the enzyme caspase-3), with the PS and quencher attached at opposing ends; and (4) a folate delivery vehicle, for targeting cancer cells that overexpress the folate receptor. This nanocarrier enters cells via a folate delivery pathway, and when activated by light, the PS produces singlet oxygen molecules that destroys the mitochondrial membrane and triggers apoptosis through the activation of caspase-3. Activated caspase-3 also cleaves the peptide linker between the PS and the quencher, restoring the PS’s intrinsic fluorescence and thus indicating those cells dying by apoptosis. Thus, the TaBIAS both induces apoptosis and also visualizes the event, using its own NIR fluorescence.

FIGURE 18.5 Schematic of the structure and function of a targeted photodynamic therapy theranostic agent with a built-in apoptosis sensor. This construct consists of a photosensitizer, a caspase-3 cleavable peptide sequence, a fluorescence quencher, and a folate-targeting vehicle. The construct accumulates preferentially in cells overexpressing folate receptors. Once activated by light, the PS produces singlet oxygen to trigger apoptosis. In the process, this leads to activation of caspase-3, which cleaves the peptide linker between the PS and the quencher, restoring the fluorescence of the PS and thus identifying those cells undergoing apoptosis using NIR fluorescence imaging. (Reprinted with permission from Stefflova, K., Chen, J., Marotta, D. et al., Photodynamic therapy agent with a built-in apoptosis sensor for evaluating its own therapeutic outcome in situ, J. Med. Chem., 49(13), 3850–3856. Copyright 2006 American Chemical Society.)

Mice bearing both negative and positive folate receptor tumors on contralateral sides were injected intravenously with the TaBIAS. A significantly greater post-PDT increase in fluorescence was observed in the folate receptor positive side of the tumor, confirming the targeting and apoptotic-reporting function of the photo-triggered theranostic agent.

18.7 CONCLUSIONS AND FUTURE OUTLOOK

Nanotechnology offers a variety of new materials for theranostics. As described in this chapter, various nanosized theranostic agents are being investigated, to combat life-threatening human diseases. However, despite this encouraging progress, there is currently no commercially available NP-based theranostic agent. Although many NP platforms have shown promise and present a number of advantages, extensive preclinical studies are needed to demonstrate their safety and efficacy. Some of the nanosized materials also suffer from a number of specific problems that are still without proper solutions. A particular issue is the biocompatibility of many of these synthetic constructs, as well as their limited biodegradation and clearance in vivo; this will remain a significant challenge, particularly for clinical translation and FDA regulatory approval. For example, most inorganic nanosized materials are nonbiodegradable and have a long retention half-life in the body. Although preclinical studies have shown these materials have low cytotoxicity and an absence of acute toxic side effects, prolonged retention in the body may induce toxic side effects many years later.

Current research activities in theranostics have placed a considerable emphasis on the design of ever-increasingly sophisticated and complex structures. However, the field would also benefit considerably from addressing toxicological and biocompatibility issues, as well as the relevant pharmacokinetic hurdles (absorption, distribution, metabolism, and elimination) of these agents. In particular, their degradation and clearance in vivo need to be resolved, in order to move forward and translate these systems into commercial products. Furthermore, the development of cost-effective synthesis of theranostics would accelerate the clinical translation of this new therapeutic paradigm.

Many nanosized theranostics are designed to target malignant tumors. A significant challenge in cancer treatment is tumor heterogeneity. A goal of theranostics is to help physicians identify the populations responding to the available therapies and find new therapies for the nonresponding populations. The design of safe and effective new theranostic modalities that could detect and differentiate tumor aggressiveness earlier, identify tumor responsiveness to therapies, and guide and inform therapeutic optimization, would bring us closer to the goal of curing life-threatening human diseases.

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