The Targeted Delivery Of Therapeutic Molecules

Published Date: 02 Nov 2017

Muthunarayanan Muthiah a,b, In-Kyu Park a,b,* Chong-Su Cho c,**

aDepartment of Biomedical Science, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-746, South Korea

bClinical Vaccine R&D Center, Chonnam National University Hwasun Hospital, Jeonnam 519- 763, South Korea

cDepartment of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea

*Corresponding author: Tel.: 82-61-379-8481 fax.: 82-61-379-8455; e-mail:[email protected]

**Corresponding author: Tel.: 82-2-880-4868; fax.:82-2-875-2494; e-mail:[email protected]

Abstract

Carriers for the delivery of therapeutic molecules and imaging agents have advanced in the field of nanobiotechnology. Most carriers are based on polymeric molecules. Biocompatible polymers have been developed to deliver molecules in a cyto-compatible way. Targeting of biocompatible polymers to a specific region is of interest to many researchers because it would reduce unwanted side effects. This chapter focuses mainly on modifications introduced to polymers to improve their biocompatibility, delivery efficacy, imaging, and targeting. The imaging of carriers and associated molecules is important to determine the fate, distribution, and pharmacokinetics of the therapeutic molecules in the system after delivery. Natural and synthetic polymers employed for the purpose of safer drug delivery and imaging are discussed. Modifications of these polymers with carbohydrate based molecules for targeting are also reviewed to shed light on current research in this area.

Key words: Imaging, contrast agent, nanoparticles, targeting, and theranostics.

1. Introduction

Imaging involves capturing a subject with an electronic device and further processing it for visualization through a display unit. The field of imaging is multidisciplinary. It demands a knowledge of math, science, and technology [1]. Bioimaging technology, as its name implies, is utilized for biological purposes such as visualizing morphological or pathological changes in the body during disease, with the help of different instruments and techniques [2]. Molecular imaging is a more advanced technique because it is able to detect changes in the body at a molecular level. Molecular imaging enables the visualization, characterization, and quantification of biological processes at cellular and subcellular levels within subjects [3]. There are two types of commonly available molecular-genetic imaging based on reporter genes and fluorescent-labeled probes. The expression level of a therapeutic gene can be evaluated by monitoring reporter gene expression. A plasmid constructed to express therapeutic and reporter genes simultaneously and that recombinant plasmid will be transfected into target cells. The therapeutic protein expression can be confirmed with the reporter gene expression, since the transcription and translation of both the genes occur simultaneously. By assaying reporter protein activity, the expression of the therapeutic gene can be deduced. Reporter gene expression assays include β-galactosidase, alkaline phosphatase, luciferase, and green fluorescent protein (GFP) expression detection [4]. Fluorescence imaging uses a fluorophore, which is excited by an external light source with a wavelength a little shorter than that of the emitted light. The respective optical filters in suitable range are used to select excitation and emission wavelengths for fluorescent imaging, although they are only applied to emitted light for bioluminescent imaging [5]. The imaging agents are the molecules used for enhancing the signal of the subject with respect to the surroundings. There are many imaging modalities available for clinical diagnosis and research purposes. The contrast agent for each imaging technique varies according to the requirement of the mode used for imaging, for example, metal oxides used for MR imaging, organic dyes used for fluorescent imaging, and radioactive materials used for PET imaging.

In this chapter, natural and synthetic polymers employed for the purpose of safer therapeutic molecules and imaging are discussed. Modifications with carbohydrate based molecules for targeting are also covered to shed light on current research in this area.

2. Classification of imaging agents

In general, imaging agents can be divided into two broad categories: endogenous and exogenous.

2.1. Endogenous agents

Luciferase from the firefly Photinus pyralis (Fluc) is the most widely used bioluminescence reporter gene. When Fluc acts on luciferin, luciferase converts chemical energy into photons, without the help of external light. GFP has been used to track specific molecules within cells[6]. In vitro, fluorescent protein indicators can be designed to track the distribution and localization of specific proteins or to visualize the responses of cells and tissues to biological events. This fluorescent pigment is covalently attached to the protein during synthesis. Consequently, the expression of the gene that encodes for GFP leads directly to the appearance of the green fluorescent signal, which is determined by the position of the mature protein. A drawback of GFP is its low emission wavelength (λ=510 nm), which overlaps with the autofluorescence of many tissues [7]. Bioluminescence can be used to overcome some of the issues encountered with endogenous fluorophores. In bioluminescence imaging, a substrate (typically luciferin) is administered to an animal that has been designed to transport luciferase. When luciferase is oxidized, it emits light, thus enabling detection when the substrate and enzyme meet [7].

2.2. Exogenous imaging agents

Exogenous agents range from simple dyes used for colorimetric contrast to sensitive fluorescent probes and beyond. Examples of common exogenous agents are lanthanide chelates, organic fluorophores used in fluorescence imaging, and gadolinium chelates and superparamagnetic iron oxide used in MRI [8]. There are a number of limitations associated with the use of conventional contrast agents such as organic dyes and gadolinium chelates [9].

2.2.1. Issues with conventional contrast agents

Organic fluorescent dyes are the most commonly used fluorophores. Dyes such as fluorescein isothiocyanate and carboxyfluorescein diacetate succinimidyl ester have been used in various biological applications [10]. The main limitation of using organic dyes is their susceptibility to rapid photobleaching because they cannot fluoresce continuously for extended periods. Thus, they are unsuitable for extended periods of bioimaging observations. Organic fluorophores are not well suited for simultaneous multicolor imaging applications. Most organic fluorophores have a relatively broad emission spectrum that can easily overlap with the emission of other fluorophores. Moreover, each fluorophore can be optimally excited only by a defined wavelength of light. Therefore, it is usually necessary to use as many excitation sources as types of fluorophores [11]. Emission/excitation is often susceptible to changes in the local chemical environment (e.g., pH, interacting ions, etc.). Emission from dyes can overlap with autofluorescence from tissues. Autofluorescence in tissues is due to the presence of low amounts of fluorophores such as nicotinamide, flavins, collagen, and elastin. The presence of these molecules generates background fluorescence. This must be overcome when looking for signals derived from organic dyes, which unfortunately also often fluoresce in the same region. This is a common shortcoming of the majority of dyes that exhibit fluorescence in the visible region [12]. Thus, the use of exogenously administered fluorochromes that fluoresce in the Near infrared (NIR) region, e.g., cyanine dyes, has increased in fluorescence imaging, although most conventional dyes emitting fluorescence light beyond ∼850 nm suffer from low quantum yield (low brightness) and poor photostability [13].

2.2.2. Advantages of nanoparticles as imaging agents

Nanoparticles have unique physico-chemical properties that make them beneficial for use as endogenous contrast agents. Coating nanoparticles with biocompatible polymers decreases their toxicity, thereby making them promising candidates as imaging contrast agents. Another important aspect of nanoparticles is that their surface to volume ratio increases as the spherical particles decrease in diameter. When the ratio is high, it allows for greater surface functionality, which can be tuned with targeting agents for specific in vivo imaging [14].

Compared with larger sized particles, imaging agents based on smaller ones have a wider distribution in vivo because they are unrestricted by biological barriers affecting the larger particles [15]. The ease of functionalization with nanoparticles has been utilized for conjugation with targeting moieties to reduce nonspecific uptake, increase specific imaging, and enhance contrasting properties [16]. Nanoparticles can be prepared with two different contrast agents at the same time. Thus, multimodality imaging is possible with a single particle administration [17, 18]. Numerous studies are ongoing to develop dual contrast agents with targeting capabilities [17-20].

The advantage of using nanoparticles as contrast agents is that they can be stable under both in vitro and in vivo conditions [21]. Nanoparticulate contrast agents are resistant to photobleaching, which is a serious problem while using fluorescent dyes for imaging. Nanoparticles have a high quantum yield and high absorbency when compared with traditional imaging agents [22]. They are also resistant to metabolic disintegration, which helps the biocompatibility of the nanoparticles. The dispersibility of nanoparticle imaging agents, except hydrophobic nanoparticles, is enhanced in the biological environment. Emission in the NIR 700–900 nm range is possible with nanoparticles. This helps to overcome autoflouresence emitted by proteins in the body [23].

2.2.3. Nanoparticle contrast agents used for imaging

Quantum dots (QDs), gold nanoparticles, and iron oxide nanoparticles are employed as contrast agents. QDs are used in optical imaging, gold nanoparticles are employed in optical and photoacoustic imaging, and super paramagnetic iron oxides (SPIONs) are deployed in MRI [24]. The properties and advantages of each type of nanoparticle are discussed below.

2.2.3.1. Quantum dots (QDs)

QDs are semiconductor nanocrystals that luminesce due to quantum confinement effects. QDs are considered a new class of biomarkers, which replace fluorescent dyes previously used in imaging. The luminescence property of QDs aids cellular tracking and labeling [25]. The atoms are confined in all three dimensions. This restricts the size of the dots to between 1 and 10 nm and leads to the quantum mechanical behavior of the particles. The fluorescence of semiconductor nanocrystals is the result of radiative recombination of an excited electron-hole pair. The excitation and emission depend on the size of the nanocrystal because the optical properties are dependent on the size of the QDs [26]. QDs have to be capped with a protective layer of insulating material because they are susceptible to photobleaching. The insulating material is aimed at preventing the photo-oxidation of the QDs. It should be translucent, nonemissive, and similar to the core to confine the excitation to the core [27]. The emission of QDs can be tuned in a wide range by changing the composition and size of the core of the material. QDs have a broad excitation spectrum and a narrow emission spectrum. This helps to reduce spectral overlap and to distinguish multiple flourophores when used simultaneously. A single excitation wavelength can excite QDs of different colors at the same time due to their broad excitation spectrum. This is an important consideration for a imaging agent when compared with currently used organic dyes, which have narrow excitation and broad emission [28]. QDs escape metabolic degradation due to their inorganic nature and passivating coating, which also plays a significant role in their resistance to photobleaching. QDs can be conjugated to linkers by covalent bonding, chelation, and electrostatic interaction. Carbodimide- mediated conjugation of QDs to amine or carboxyl groups of proteins is the most commonly used bioconjugation procedure [29].

2.2.3.2. Gold nanoparticles

Gold nanoparticles are highly convincing as contrast agents for optical imaging of biological samples [30]. Gold nanoparticles have been already used as immunochemical probes for ex vivo applications and as adjuvants for radiotherapies [31]. Gold’s strong optical resonance generated by surface plasmons makes it a better contrast agent than other metal nanoparticle based agents [30]. Surface plasmons are localized excitations of conduction electrons emitted in response to specific wavelengths of light. Gold nanoparticles have been synthesized in different nanostructures with tunable plasmon resonance modes, with resonance ranging from visible to near infrared wavelengths. Gold nanoparticles with plasmon resonance in the NIR range between 750 and 1300 nm are beneficial for bioimaging because shorter wavelengths are extinguished by hemoglobin and longer wavelengths are attenuated by water [31]. Gold nanoparticles can strongly scatter or absorb light. The magnitude of light that is scattered or absorbed is largely dependent on the structure and size of the nanoparticles. Gold nanostructures with large scattering cross-sections can be ideal for enhancing the contrast in optical coherence tomography. Gold nanostructures with large absorbance cross-sections can be used to enhance the contrast in photoacoustic imaging [15, 32]. Gold nanoparticles can also convert light energy into heat energy. Therefore, they can also be employed in photothermal therapy [33].

2.2.3.3. Super paramagnetic iron oxide nanoparticles (SPIONs)

Super paramagnetic iron oxide nanoparticles (SPIONs) can enhance the proton relaxation of specific tissues. Thus, they are utilized as MR contrast agents in clinical diagnosis. Due to their super paramagnetic properties, they do not behave as permanent magnets in the absence of external magnetic fields, although they will quickly respond to the presence of an external magnetic field [34]. This important property of iron oxides has enabled them to serve as contrast agents for more than two decades.

SPIONs possess unique magnetic properties, with strong shortening effects under longitudinal relaxation and transverse relaxation pathways. T1 relaxation of SPINs occurs due to spin-lattice relaxation (i.e., energy exchange between the spins and the surrounding lattice), which re-establishes the thermal equilibrium. Radio frquency (RF) energy is released back into the surrounding lattice when the spins travel from a high energy to a low energy state. T2 relaxation of SPINs is the result of spin-spin interactions, which occur when the spins get out of phase. Transverse magnetization decay occurs as a result of a cumulative loss in the phase during spin-spin relaxation when the transverse relaxations are temporary and random [35].

SPIONs shorten the T2 relaxation of neighboring tissues. This results in a decrease in the signal intensity in MR images. As reported previously, SPIONs in the nanoregime improve biodistribution compared with conventional chemical agents [36]. Surface modification of SPIONs with targeting moieties aids specific and early diagnosis with MRI. Moreover, SPIONs can be conjugated with drugs to facilitate monitoring of the distribution, release, and tracking of therapeutic molecules[37, 38]. In addition, they can be exploited to transport a drug to a specific region via magnetofection, which attracts the drug to a specific location with the help of strong magnets placed near the target site [39].

2.2.3.4. Problems with naked nanoparticles

The bare nanoparticles without any coating or stabilizing agent (naked nanoparticles) usually show aggregation in water, chemical instability in air, and a lack of biodegradability in the physiological environment. Naked nanoparticles undergo nonspecific interactions with serum proteins and tend to agglomerate in vivo due to hydrophobic-hydrophobic interactions between the particles when exposed to a physiological environment [21, 22, 40]. The nanoparticles are also rapidly eliminated from the circulation due to this aggregation, following adsorption of the serum proteins onto the surface of the nanoparticles. In addition, these particles circulate inside the body without any specific interaction with the organs or tissues. The surface of the nanoparticles must be hydrophilic for in vivo applications because they have to be stable for a prolonged time inside the body’s circulatory system [41].

3. Surface modification of nanoparticle contrast agents

QDs undergo photobleaching and photo-oxidation. To avoid these problems, a coating material is necessary. Bare SPIONs are hydrophobic. They can aggregate in the biological environment and vary in their contrasting capabilities [42]. Coating materials play an important role in the stabilization of aqueous nanoparticle suspensions, as well as in their further functionalization. Surface modification of nanoparticles with various materials can effectively render them water soluble and improve their stability under physiological conditions. There are two approaches available for coating nanoparticles: ligand addition and ligand exchange. With the ligand addition mechanism, polymers are physically adsorbed on the surfaces of the nanoparticles due to electrostatic and hydrophobic interactions and hydrogen bonding [43]. Polymers with functional groups (i.e., hydroxyl, amine, carboxyl, etc.) at the periphery of nanoparticles readily absorb to the surface of the nanoparticle. Surface modification of prepared nanoparticles is an essential step for safe clinical applications. Tailoring the surface of the nanoparticles with polymers and other materials helps to improve their stability, surface charge, functionality, and targeting capability. The ideal polymer for a nanoparticle coating should have a high affinity for metal oxides, without stimulation of the immune system or antigenicity. The biocompatibility of the coating is a vital element when considering in vivo applications. When hydrophobic nanoparticles are injected into the bloodstream, they are surrounded by plasma proteins (hydrophobic surface) in a process called opsonization [44]. Once the nanoparticles enter the physiological environment, they interact with hydrophobic surfaces, resulting in aggregation due to hydrophobic-hydrophobic interactions. Therefore, hydrophobic nanoparticles are prone to opsonization and tend to be cleared immediately by the mononuclear phagocytic system (MPS). On the other hand, if hydrophobic nanoparticles are coated with hydrophilic polymers, the interaction of the nanoparticles with the plasma proteins can largely be avoided. Thus, a hydrophilic coating on the nanoparticles will increase the in vivo circulation by reducing uptake by the MPS [45]. It will also confer stability and functionalization, enabling further drug, gene, or imaging agents to be conjugated. These properties make nanoparticles multifunctional, allowing them to serve both as contrast agents and drug carriers with a particular specificity. Hydrophilic materials used for nanoparticle coatings include proteins, polysaccharides, lipids, and synthetic polymers. The most commonly used biocompatible coating materials are polyethylene glycol (PEG), dextran, polyvinyl alcohol (PVA), chitosan, pullulan, alginate, gelatin, and polyvinyl pyrrolidone (PVP) [46, 47].

3.1. Chitosan

Chitosan is found in abundance from sea sources and is known to be biocompatible, hydrophilic, biodegradable, nonantigenic, and nontoxic [48]. Functional groups present on the chitosan form complexes with metal oxide surfaces, making the nanoparticles hydrophilic, biocompatible, and stable. The presence of positively charged amino groups in chitosan facilitates interactions with negatively charged nucleic acids, which can be delivered along with the imaging agent. Furthermore, chitosan is known to facilitate particle movement across cellular barriers and transiently open tight junctions between epithelial cells `[49, 50]. This polymer has been used to coat SPIONs to provide a better contrast agent for MRI.

Hong et al. prepared chitosan-coated ferrite nanoparticles (CFNs) as an MRI contrast agent and injected them into mice [51]. In the resulting MR images, the part of the liver where the agent was injected was darker than the same area of the liver in mice that had not been injected. The darkening is due to faster T2 relaxation of nuclear spins in the liver because of the uptake of ferrite nanoparticles by Kupffer cells (liver macrophage cells). When they injected the CFNs into a hepatoma model, signal intensity at the site of the hepatoma showed little change due to a lack of Kupffer cells or a reduced number of these cells in hepatomas. Consequently, hepatoma sites can be easily identified using ferrite nanoparticles as a contrast agent. Ge et al. prepared nanoparticles coated with modified chitosan possessing a magnetic oxide core and a covalently attached fluorescent dye [52]. SPIONs labeled with fluorescent chitosan enabled direct imaging and localization in living cells. The labeled cells were observed in a 1.5-T MR imager with detectable cell numbers of about 104 in vitro. Magnetic fluorescent nanoparticles can serve both as MR contrast agents and as optical probes in intravital fluorescence microscopy.

3.2. Polyethylene glycol (PEG)

PEG is widely used to enhance the aqueous solubility of hydrophobic imaging agents. A PEG coating helps to minimize the uptake of reticulo endothelial system (RES), and it increases the circulation time of the nanoparticles without generating an immune interaction. It minimizes nonspecific interactions with the body, which further aids tumor accumulation of PEG due to enhanced permeability and retention [44]. Once the nanoparticles are coated with PEG, they act as a good spacer for the attachment of different biomolecules. If ligand-like proteins or antibodies are attached to PEG, the accumulation of the nanoparticles will be more specific to the region of interest, while sparing normal cells[53].

Mahmoudi et al. synthesized SPIONs coated with cross-linked polyethylene glycol-co-fumarate (PEGF) [54]. They observed enhanced aqueous stability of the PEGF-coated SPIONs due to the hydrogel property of PEGF, which enables it to absorb water and decrease the density of the core-shell nanoparticles. In addition, the hydrogel coating is able to take up and release drugs in response to physical and chemical changes in the environment. MRI of the drug-loaded PEGF was possible due to the presence of the SPIONs at the core. The drug loading was efficient due to the cross-linking of the PEG with fumarate. This combination is best suited to theranostic agents, which are described later in this chapter (Section 6).

Yu et al. exposed porcine aortic endothelial cells to 5 and 30 nm diameter iron oxide nanoparticles coated with the polysaccharide dextran or with PEG [55]. In both 2D and 3D cell culture studies, they found that bare nanoparticles decreased cell viability and that dextran- and PEG-coated nanoparticles did not show any reduction in cell viability. PEG-coated contrast agents are expected to be more biocompatible under in vivo conditions and to prevent nonspecific interactions of the contrast agents with proteins in the body.

3.3. Dextran

Dextran has been successfully used for various in vivo applications [56, 57]. Dextran-coated SPIONs are commercially available clinical contrast agents for MRI, and they have been shown to possess cancer nodal staging capabilities [58]. To improve stability and functionality, a carboxymethyl group was added to the coating, and dextran was cross-linked with epichlorohydrin to form cross-linked iron oxide nanoparticles (CLIOs) to further enhance their stability. The CLIOs had better stability compared with the dextran-coated SPIONs containing the carboxymethyl group, without undergoing any alterations in their sizes. These functionalized ligand-conjugated CLIOs have been extensively evaluated for MRI applications [59].

In a study of SPION ferumoxytol administered to prostate cancer patients, Harshingani et al. showed accumulation in benign lymph nodes, with changes in the signal to noise ratio (SNR) [60]. They found only a slight change in the SNR ratio at malignant lymph nodes, suggesting that nodal staging can be detected in MRI with the help of ferumoxytol in an efficient and safe manner.

Josephson et al. synthesized TAT-cross-linked and dextran-coated SPIONs and studied their cellular localization in human lymphocytes, natural killer cells, and HeLa cells [61]. Immune histochemical staining revealed that the CLIOs were located in the nucleus and not in the cytoplasm. Moreover, they reported that the CLIO-labeled cells were highly magnetic and detectable by MRI. They were also retained on magnetic separation columns, demonstrating that cross-linked SPIONs are more stable than noncross-linked ones. The above studies reveal that dextran-coated SPIONs are biocompatible and stable contrast agents for MRI.

3.4. Polyvinyl alcohol (PVA)

PVA is a biocompatible and water-soluble material used for coating SPIONs. PVA can resist protein adsorption and cell adhesion and also has high biocompatibility. PVA has already been applied for tendon repair, contact lenses, ophthalmic materials, drug delivery, and various other biomedical applications [62].

Fink et al. prepared carboxyl-modified (CM) and thiol- and amino-modified PVA and tested their properties in human melanoma cells [63]. They observed that the iron content was always below the detection limit in cells exposed to PVA-SPIONs, CM-PVA SPIONs, or thiol-modified PVA SPIONs. However, in cells exposed to amino-SPIONs, the cellular iron content was increased after 24 h of continuous exposure, and it was dependent on the amount of the treated SPIONs. The results suggest that presence of the amino groups in the PVA is necessary for improving the cellular uptake of SPIONs.

Kayal and Ramanujan coated SPIONs with different percentages of PVA and loaded them with doxorubicin for anticancer drug delivery [64]. They then studied the doxorubicin loading and release profiles of the PVA-coated SPIONs. The results showed that up to 45% of the drug adsorbed was released in 80 h and that the drug release pattern involved the Fickian diffusion-controlled process, suggesting that SPIONs can be used for imaging and tracking the loaded drug along with its polymer coating.

3.5. Polyvinyl pyrrolidone (PVP)

PVP has been used in various biomedical applications because it is biocompatible, aqueous, soluble, and possesses a neutral charge. The PVP coating has been achieved by covalent interactions in most work. This increased the stability of SPIONs in physiological medium [65].

Lee et al. prepared PVP-coated iron SPIONs to study their efficacy as a MRI contrast agent [66]. Macrophage uptake of large-core PVP-SPIONs was higher than that of commercially available contrast agent Feridex uptake. T2∗-weighted MRI results also indicated that PVP-SPIONs were better than Feridex as a T2∗ negative contrast agent for MRI. The signal intensity drop was enhanced in a large-core PVP-SPION-treated rabbit liver parenchyma model compared to a Feridex-injected animal model.

Arsalani et al. grafted PVP onto SPIONs by surface-initiated radical polymerization [67]. They reported that the SPIONs exhibited increased and stable water dispersibility for several months. In addition, the PVP-grafted SPIONs showed improved T2∗ relaxivity compared with dextran-coated SPIONs already approved for clinical use. They attributed the improvement to the grafting of the polymer on the metal surface. This enables it to survive longer, without becoming separated from the contrast agent even under in vivo conditions. This aids the biocompatibility of the nanoparticles, which otherwise would lose their importance as safe contrast agents in clinical usage.

4. Targeted imaging

Targeted imaging is very important to avoid nonspecific accumulation of the contrast agent and to reduce the amount of contrast agent required to show the enhancement. The interaction of the contrast agent with proteins in the body during circulation reduces the amount of contrast agent reaching the target site, thereby influencing the contrast enhancement of the site [68]. Passive accumulation of contrast agents in tumors due to enhanced permeation and retention is comparatively less than the accumulation in other organs such as the kidney and liver. Therefore, active targeting with specific targeting agents such as antibodies, peptides, proteins, and carbohydrates is necessary [69]. In this section 4, we focus on carbohydrate-based targeted imaging agents.

4.1. Hepatocyte-targeted imaging

Asialoglycoprotein receptors (ASGP-Rs) found on the cell surfaces of hepatocytes interact specifically with the galactose moiety [70]. When galactose or N-acetylgalactosamine residues are attached to the surfaces of contrast agents, they are taken up specifically by hepatocytes, and the signal specifically in that region is enhanced.

Yoo et al. coated SPIONs with polyvinyl-benzyl-O-β-D-galactopyranosyl-D-gluconamide (PVLA) to target hepatocytes [71]. PVLA contains galactose moieties, which help it to recognize the ASGP-R present on the surfaces of hepatocytes. Due to its specificity, significant uptake of the PVLA-coated SPIONs in the liver was observed within an hour. The preferential accumulation of the PVLA-coated SPIONs in the liver resulted in an enhanced signal drop in the liver compared with PVP-coated SPIONs (Fig. 1), indicating that targeted contrast agents largely aid faster diagnosis than nontargeted contrast agents.

Selim et al. prepared lactobionic acid (LA)-coated SPIONs to target hepatocytes [72]. Uptake of the LA-coated SPIONs by the hepatocytes was enhanced compared with uptake by unmodified and maltotrionic acid-modified nanoparticles. It can be expected that LA-coated SPIONs will be used as a specific recognition marker for hepatocytes to aid specific imaging of the liver for diagnostic purposes.

4.2. Immune cell targeting

Mannan is recognized by mannose receptors of antigen-presenting cells (APCs) and reticuloendothelial cells mainly residing in normal lymph nodes [73]. The circulating APCs can also opsonize the circulating contrast agent coated with mannan and then migrate to the lymph nodes. To investigate mannose receptor recognition on macrophages, Yoo et al. coated mannan on the surface of SPIONs [74]. The uptake of the mannan-coated SPIONs(mannan-SPIONs) was enhanced in macrophages when compared to PVA-coated SPIONs. In vivo MR images of the mannan-coated SPIONs indicated that the signal intensity significantly dropped in the liver region. Through Prussian blue staining, Yoo et al. found that the uptake of the mannan-SPIONs was selective in Kupffer cells compared with PVA-SPIONs, indicating that mannan-SPIONs were mainly accumulated by macrophage-targeted uptake.

In another study, glucan was coated on SPIONs to image the metastatic liver because glucan is recognized by the dectin-1 receptor present on the immune cells in the liver [75]. When the glucan-coated SPIONs were injected in vivo, the Kupffer cells in the metastatic liver took up the contrast agent. Uptake of the contrast agent by the tumor was not found because the metastatic tumor region in the liver is devoid of immune cells. The immune cell rich region in the periphery of the tumor appeared dark during MRI, and this helped in distinguishing the normal tissue from the cancerous tissue (Fig. 2).

Mannan-coated SPIONs have also been targeted to lymph nodes via immune cells present in the nodes [76]. After a local injection of mannan-coated SPIONs in normal mice, the accumulation of the system was found in nearby lymph nodes within an hour, and this was detected via MRI. This system can be utilized to diagnose metastatic tumors in lymph nodes. When the mannan-coated SPIONs were injected in the lymph node metastatic model, they preferentially interacted with the immune cells present in the nodes around the tumor region. Therefore, the region around the tumor was dark during the MRI due to the uptake of the mannan-coated SPIONs by the immune cells in the lymph nodes [76] (Fig. 3). In the study, the LD 50 value for systematic injection of the contrast agent was 44 mg/kg. To improve the biocompatibility, a carboxyl group was introduced to the mannan in another study [77]. The LD 50 value of the mannan-coated SPIONs including the carboxyl group increased up to 80 mg/kg, indicating the enhanced biocompatibility of the contrast agent. In this study, the in vitro and in vivo targeting capabilities of mannan were retained, and faster accumulation and a signal drop remained even after carboxylation.

5. Multimodality imaging

Using multimodality imaging, the weaknesses of one modality can be offset by the strengths of another modality (Table 1). A combination of different modalities is required to diagnose malignancy at an early stage. Nanoparticles such as QDs, SPIONs, and gold nanoparticles have different properties necessary for multimodal imaging [19]. Therefore, multimodal imaging can be achieved with a single nanoparticle contrast agent or with two different particles in a single delivery vehicle. Positron emission tomography (PET) / computed-tomography (CT) and PET/( magnetic resonance imaging) MR are common examples of multimodality imaging. PET can provide functional information, and CT/MR can provide anatomical information [78].

Bouchard et al. prepared a nanoconstruct consisting of ferromagnetic (Co) particles coated with gold for MR/photo acoustic tomographic (PAT) imaging [79]. The gold coating enhances the PAT imaging because its absorption matches the near infrared laser excitation used in PAT. Moreover, the gold coating aids biocompatibility, and the unique shape of the particles enables optical absorption over a broad range of frequencies. The combination of MRI and PAT into a single delivery molecule offers the advantage of edge detection and contrast-based volume imaging at the same time.

Misri et al. conjugated the 111 In-labeled antimesothelin antibody to SPIONs [80]. This 111 In-labeled antimesothelin antibody coated SPIONs contrast agent used in single photon emission computed tomography (SPECT) imaging and in MRI. SPECT imaging facilitates noninvasive determination of the in vivo biodistribution of radiotracers at picomolar concentrations. It is combined with SPIONs to obtain anatomical information through MRI. The antimesothilin antibody coated with contrast agents aids specific targeting and imaging of mesothelin-expressing tumors. Combining SPECT and MRI contrast agents in a single delivery vehicle with the targeting moiety offers a powerful dual modality diagnostic tool for early diagnosis of tumors.

6. Nanotheranostics

Theranostics involves simultaneous diagnosis and therapy within a single formulation (Fig. 4). Many studies have combined a drug and an imaging agent in a single delivery molecule for imaging and therapy [81, 82]. They can be coformulated in the core of liposomes and polymersomes because most drugs and imaging agents are hydrophobic. The contrast agents can also form the core of the particle, which can be coated with polymers. The drug then interacts with the polymers on the surface. The drug can be released at the target site, and the imaging agent can be used to track, image, and quantify the drug [83]. The development of targeted theranostic agents has increased in importance because accumulation in normal regions is more toxic, and many constituents are present in the theranostic nanoparticles.

Many studies in the literature have described the use of theranostic agents with chemical drugs and imaging agents [83, 84]. Thus, we focus here on theranostic agents that deliver biological products (siRNA, miRNA, and proteins). Contrast agents have been loaded onto cationic liposomes or polymersomes, and genes have been attached to the cationic surface for gene therapy monitoring in an efficient manner [85]. Gold nanoparticles, QDs, silicon, and SPIONs have been utilized extensively for gene delivery[86]. Numerous studies have already reported siRNA and plasmid DNA delivery with these contrast agents [87, 88].

Hao et al. prepared polyvalent oligonucleotide functionalized gold nanoparticles for the delivery of tumor suppressor miRNA 205 and oncogenic miRNA 20a [89]. These oligonucleotide functionalized gold nanoparticles were transported to the cells without the use of a cationic cocarrier. After treatment with miRNA 205 mimics -gold nanoparticle, the cellular survival of the prostate cancer (PCa) cells was drastically reduced, and their migration capabilities ceased. Hao et al. also used oncogenic miRNA 20a with gold nanoparticles and tested its target binding capabilities in relation to E2F1 and the reduction of the expression of PTEN.

Yoon and Natarajan et al. delivered miRNA491 to induce apoptosis in human breast cancer cells (HT3477) using QDs, gold nanoparticles, and HBT3477 targeting Mab [90]. They also developed novel immunonanoparticles linked to miRNA to track, target and selectively deliver the construct into the cancer cells in vivo. Their molecular assembly consisted of QD 655, breast cancer targeting ChL6, and apoptosis-inducing miRNA attached to gold nanoparticles.

Conclusion

The development of nanoparticles as imaging agents and therapeutic molecule carriers has gained importance in the recent past. The biocompatibility of these components has been improved by coating them with biocompatible polymers, which have also played a significant role in increasing their stability and in vivo circulation. In specific targeting and imaging, carbohydrate-based moieties have demonstrated promise by exerting effects while sparing normal tissues For the accurate detection of tumors, multimodality imaging has led to advancements in the field of nanoparticulate contrast agents. Nanotheranostic agents are also now being developed that can fulfill the role of imaging agent and therapeutic agent within a single delivery vehicle, thereby providing diagnosis and therapy simultaneously.