Near-Infrared Contrast Agents for Bone-Targeted Imaging

Abstract

Background:

For the bone-specific imaging, a structure-inherent targeting of bone tissue recently has been reported a new strategy based on incorporation of targeting moieties into the chemical structure of near-infrared (NIR) contrast agents, while conventional methods require covalent conjugation of bone-targeting ligands to NIR contrast agents. This will be a new approach for bone-targeted imaging by using the bifunctional NIR contrast agents.

Methods:

The goal of this review is to provide an overview of the recent advances in optical imaging of bone tissue, highlighting the structure-inherent targeting by developing NIR contrast agents without the need for a bone-targeting ligand such as bisphosphonates.

Results:

A series of iminodiacetated and phosphonated NIR contrast agents for the structure-inherent targeting of bone tissue showed excellent bone-targeting ability in vivo without non-specific binding. Additionally, the phosphonated NIR contrast agents could be useful in the diagnosis of bone metastasis.

Conclusion:

By developing bone-targeted NIR contrast agents, optical imaging of bone tissue makes it very attractive for preclinical studies of bone growth or real-time fluorescence guided surgery resulting in high potential to shift the clinical paradigms.

Introduction

Near-infrared (NIR) fluorescence imaging is a highly sensitive and proven method, recently has been applied for preclinical and clinical studies. Real-time in vivo optical imaging by using NIR contrast agents provide unique possibilities for target-specific imaging of human tissues including bone [1], cartilage [2], parathyroid glands [3], nerve [4], vasculature [5–7], tissue perfusion [8, 9], lymph nodes [10–13], ureter [14–16], bile duct [17, 18], and various cancers [19–22]. The NIR contrast agents show minimized autofluorescence in aqueous solution, and high extinction coefficients resulting in non-invasive deep tissue imaging [23–30].

Recent advances in NIR fluorescence imaging of bone tissue enabled real-time intraoperative detection of bone metastasis, bone growth, and tissue microcalcification [31–33]. As first introduced in 2001 [1], the conventional approach to NIR fluorescence imaging of bone tissue required the covalent chemical conjugation of bone-targeting ligands, including calcein, tetracycline and bisphosphonates to NIR dyes for creating a bifunctional contrast agent [34–37]. Tetracycline and its derivatives as divalent cation chelating agents have been used to treat osteoporosis and bone metastasis, and labeled for fluorescent imaging, because these readily bind hydroxyapatite, inhibiting osteoclast resorption of bone matrix [38–40]. Additionally, bisphosphonates have also been used as bone-targeting drugs in a variety of contrast agents for the detection and treatment of bone diseases [41–46], because of their high binding affinity toward bone minerals and significant therapeutic effects. However, their major drawback is poor solubility in organic solvents for chemical conjugation to the NIR contrast agents resulting in low yields [1, 35]. Moreover, the targeting properties of them may be affected by chemical modification, therefore, the fundamental problems still remained unsolved [19, 21, 22]. A simpler and more straightforward strategy is required for the use of NIR contrast agents for non-invasive bone tissue imaging.

In response to this unmet needs, several groups recently reported a class of structure-inherent targeting as dual imaging and targeting agents for bone-specific imaging [47–50]. These NIR contrast agents are composed of squaraine-based rotaxane and heterocyclic polymethine cyanines performing both imaging and targeting properties. They have demonstrated preferential accumulation and retention in bone tissue in vivo, with no accumulation in other normal tissues, enabling bone-specific targeting without the need for additional ligand conjugations. Furthermore, the bone-targeted NIR contrast agents show excellent stability and strong fluorescence in bone tissue after being chemically modified to improve the affinity and specificity of the molecules. Importantly, these NIR contrast agents do not cause cytotoxicity or systemic toxicity in animal models when administered in a dose range appropriate for NIR fluorescence imaging. Thus, the simultaneous imaging and targeting capabilities of bone-targeting contrast agents could be further applied for diagnosis and therapy of bone diseases. This strategy is the paradigm shift in a role of contrast agents from conventional methods needed for covalent chemical conjugation of targeting ligands and imaging agents.

In this review, we focus on the use of bone-targeting NIR contrast agents and emphasize a novel strategy of structure-inherent chemical recognition in bone-targeted imaging potential for preclinical and clinical use.

Conventional methods for NIR fluorescence imaging of bone

For the target-specific optical imaging, a traditional method for preparing targeted imaging agents requires chemical conjugation of separate targeting ligands and fluorescent agents. Several groups previously reported the development of bone-specific NIR contrast agents by conjugating commercially available NIR dyes such as IRDye78 [1], IRDye800CW [34, 35, 51], Alexa Fluor 647 [51], and ZW800-1 [37] with tetracycline derivatives or bisphosphonate drugs. Although the NIR dye conjugates showed rapid and specific binding to bone minerals in vitro and in vivo, the molecular effect of conjugated NIR dyes on bone targeting potency and in vivo performance needs to be considered. Indeed, overall molecular net charge and hydrophobicity of the NIR dye conjugates play a crucial role in the pharmacokinetics, biodistribution and clearance. Frangioni’s group previously demonstrated that physicochemical properties of the NIR dye conjugates have strong influence on the targetability, specificity and dye selection for targeted contrast agents [5, 19, 22].

Tetracycline-based bone-targeting contrast agents

Although there are many types of divalent cation chelating agents such as calcein [52, 53], DCAF (2,4-bis[N,N′-di(carboxymethyl)aminomethyl]fluorescein) [54, 55], tetracycline and its derivatives [8, 10, 11], alizarin red and its derivatives [56], xylenol orange [57], or bisphosphonate drugs [41–46], several fluorescent agents all have the spectral limitation of absorption/emission spectra shown in the visible region, which is not ideal for noninvasive fluorescence imaging. To solve this problem, as shown in Fig. 1A, Kovar et al. [36] previously reported a NIR optical bone marker for preclinical animal imaging by conjugating tetracycline derivative to commercially available IRDye800CW dye (BoneTag™ agent; BT). They demonstrated that tetracycline-labeled IRDye800CW could be used to identify a changing mineralization in bone sections from normal growing mice and an osteoporosis mouse model by comparing cortical bone in non-treated and ovariectomized mice. This result suggests that the NIR-labeled BT is effective as a general marker of bone features and an indicator of the bone mineralization and remodeling processes.

Fig. 1.

A NIR fluorescence imaging of bone tissue by using a tetracycline-labeled IRDye800CW (2 nmol of BoneTag™ agent) 24 h post-injection. White arrows in the dorsal and lateral views indicate kidney location. B Bone-specific imaging for three different types of pamidronate-conjugated NIR fluorophores (10 nmol) in mice 4 h post-injection. Arrowheads indicate cartilage tissues. Li liver.

Reproduced with permission from Ref. [36] (Copyright 2011 Elsevier Inc) and Ref. [37] (Copyright 2015 Ivyspring International Publisher)

Bisphosphonate-based bone-targeting contrast agents

Bisphosphonates have been widely used as targeting and therapeutic agents for the treatment of bone diseases such as osteoporosis and bone metastasis [41–46]. Fluorescent bisphosphonate imaging is a fast and reliable technique for visualizing site-specific delivery and retention of bisphosphonate in vivo. Since Frangioni’s group firstly reported pamidronate-labeled IRDye78 in 2001 for in vivo NIR fluorescence imaging of bone tissue [1], various types of bisphosphonate-based NIR contrast agents have been continuously explored and discussed [34, 35, 37]. Recently, Bao et al. [37] demonstrated that in vivo fate of the targeting moiety (i.e., bisphosphonates such as pamidronate) is determined by the physicochemical properties of conjugated NIR dyes, because the size of targeting ligand is smaller than imaging domain. Since bisphosphonate is a negatively charged small molecule, the optimal balance of total surface charge and hydrophobicity after conjugation to NIR dyes are of significant importance to design targeted contrast agents for bone-specific imaging and optimized in vivo performance (Fig. 1B).

Structure-inherent targeting of NIR contrast agents for bone tissue imaging

A new strategy of structure-inherent targeting is based on the incorporation of targeting moieties into the non-delocalized structure of contrast agents such as pentamethine and heptamethine indocyanines performing both targeting and imaging, simultaneously [58–64]. Using the known affinities of iminodiacetate and phosphonates for bone minerals, two different types of bifunctional contrast agents that target bone tissue without requiring the conjugation of ligands were recently reported [47, 48]. A single contrast agent could be designed to achieve selective targeting with high efficiency in bone tissue through modifying the physicochemical properties and incorporating targeting moieties into the structural domain of contrast agent. For the design of optimized contrast agents, the relationship among the physicochemical properties such as size, charge, and hydrophobicity of NIR contrast agents on in vivo targeting and biodistribution is therefore an important consideration in the development of next generation multifunctional contrast agents [21, 59–62].

Iminodiacetated NIR contrast agents for bone-targeted imaging

Based on the strategy of structure-inherent bone targeting, Harmatys et al. [47] recently reported a new structural class of bone-seeking molecular probes composed of dendritic squaraine rotaxanes. Squaraine rotaxanes exhibited intense and narrow absorption/emission bands with deep-red wavelengths of 653–677 nm in aquous solution. For the bone-targeting ability, the squaraine rotaxane probes were decorated with multiple copies of iminodiacetate groups as the bone targeting moieties. Since the iminodiacetate groups have inherently lower bone affinity than bisphosphonates by comparing Ca²⁺ association constants [65], they designed multivalent versions of tetra(iminodiacetate)-appended squaraine rotaxanes to enhance the binding affinity to bone minerals. They demonstrated that tetra(iminodiacetate)-appended squaraine rotaxane is the optimal structure for bone-targeted imaging compared to bis(iminodiacetate) and tetra(iminodipropionate) structures in terms of bone targeting potency and in vivo performance (Fig. 2). This concept suggests that multivalent iminodiacetate groups have promise as bone targeting contrast agents for various pharmaceutical applications.

Fig. 2.

A Chemical structure of the fluorescent squaraine rotaxane probe and NIR fluorescent images (B, prone; C, leg; D, supine) of skinless mice 24 h post-injection of squaraine rotaxane probe (4 nmol) and commercial probe OsteoSense 750 (2 nmol), respectively.

Reproduced with permission from Ref. [47] (Copyright 2013 ACS Publications)

Phosphonated NIR contrast agents for bone-targeted imaging

Typically, targeted NIR contrast agents reported to date for bone tissue imaging require covalent chemical conjugation of a targeting domain (i.e., divalent cation chelating agents such as calcein and tetracycline, bisphosphonate drugs, etc.) to a NIR contrast agent [34–37]. For the structure-inherent targeting of bone tissue, Hyun et al. [48] recently reported a new strategy based on incorporation of phosphonate moieties into the chemical structure of polymethine indocyanines emitting at NIR window. As the results of this concept, longitudinal NIR imaging studies in mice demonstrated that phosphonated NIR contrast agents (P700SO3 and P800SO3) remain stable in bone tissue for over 5 weeks (Fig. 3). Furthermore, the bifunctional NIR contrast agents could be utilized to conjugate therapeutic drugs that control osteoclast activity, then creating trifunctional theranostic agents. They emphasized that phosphonate groups of the designed contrast agents may increase bone strength, acting like bisphosphonate drugs, and improve bone density by preventing the loss of minerals. Therefore, this long-term stability of phosphonated NIR contrast agents in the body could be ideal for various biomedical applications in the fields of tissue engineering and regenerative medicine.

Fig. 3.

Longitudinal stability of phosphonated NIR contrast agents targeted in bone matrix. P700SO3 and P800SO3 were injected intravenously into 20 g athymic nude mice (10 nmol, 0.4 mg/kg) 5 weeks prior to imaging.

Reproduced with permission from Ref. [48] (Copyright 2014 Wiley–VCH)

NIR fluorescence imaging of bone metastasis

The major techniques for detection of bone metastasis are magnetic resonance imaging (MRI) or computed tomography (CT), but both techniques do not provide optimum quality images because of poor accuracy. In response to this unmet clinical need, a new method recently reported by Hyun et al. [31, 32] was developed to provide real-time visualization of metastatic bone tumors by using the NIR imaging system in combination with a bone-targeted NIR contrast agent (P800SO3). A single-dose intravenous injection of the P800SO3 allowed high-quality visualization of metastatic bone tumors in the intratibial and intracardiac xenograft models, respectively (Fig. 4). The most important advantage of using the P800SO3 NIR contrast agent for bone metastasis imaging is the integrated approach to the ‘structure-inherent targeting strategy’ (i.e. molecular targeting and fluorescent imaging). Thus, the smart bone-targeted contrast agents have many beneficial applications in terms of bone diseases, bone growth, tissue microcalcification, and image-guided surgery.

Fig. 4.

A Chemical structure of P800SO3, B Osteoclast-binding specificity and C targeted bone metastasis by using the bone-specific NIR contrast agent (P800SO3). MDA-MB-231 breast cancer cells were intratibially or intracardially inoculated into different groups of mice 3–5 weeks prior to imaging, respectively, followed by 10 nmol P800SO3 injected into each mouse model 4 h before imaging.

Reproduced with permission from Ref. [31] (Copyright 2017 Impact Journals LLC) and Ref. [32] (Copyright 2019 Springer Nature)

Conclusions and perspectives

Here we discussed bone-specific contrast agents that can be used for preclinical studies of bone growth or real-time fluorescence guided surgery resulting in high potential to shift the clinical paradigms. NIR fluorescence imaging is a rapid and cost-effective tool for monitoring bone diseases and to quantitatively assess bone regeneration in living tissue. It is an emerging field that allows noninvasive monitoring of biological processes in vivo, and enables testing at intervals to demonstrate how bone tissues develop and respond to bone-targeting agents.

Importantly, developing NIR fluorescent contrast agents targeted to metastatic bone tumors will lead to novel possibilities for real-time diagnosis and treatment of bone metastasis in patients. Since a bone scan provides the less accurate diagnosis of osteolytic bone metastatic tumor than osteogenic, the target-specific NIR fluorescence imaging of osteolytic lesion on metastatic tumor could help surgeons for intraoperative image-guided surgery. The future of bone metastasis imaging by using the structure-inherent targeting of NIR contrast agents will likely involve the development of increasingly more powerful contrast agents that will greatly improve diagnostic accuracy.

Current limitation preventing the potential clinical benefits of fluorescence guided surgery is the lack of FDA approved NIR contrast agents. Although a significant number of preclinical studies by using NIR contrast agents have been reported, most of them are still unavailable in clinic. For the clinical translation, the NIR contrast agents need to be considered in terms of physicochemical and optical stability, selective targetability, reliable pharmacokinetic activity, efficient body clearance, and non-toxicity. Among the considerations, toxicity is the major issue for the FDA approval, because the incomplete clearance and excretion can cause potential toxicity through biological interactions in the body. For the clinical use, rapid biodistribution and complete excretion of the injected NIR contrast agents during a certain period of time are essential for safety. Therefore, rational design of NIR contrast agents by considering molecular properties, target specificity, biodistribution, and targeting mechanism is the only way to overcome the limitations for clinical use of NIR contrast agents.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.