CHAPTER 1: Targeting Supramolecular Imaging Agents for a Wide Range of Applications

Supramolecular chemistry is a rapidly growing area of science, with its foundations based on three key concepts: fixation, co-ordination and recognition. With roots extending not only into chemistry but also biological and physical sciences, this interdisciplinary field of science involves the formation of highly complex molecular systems through the association of two or more chemical species. Through a greater understanding of the host–guest relationship, the design of highly selective host species can be achieved, increasing the variety of applications of these supramolecular systems.

Over the years, one of the main areas within the field of supramolecular chemistry has been the development of molecular sensors for the selective complexation of suitable guest species. In both chemical and biological systems, ions and molecules occur in abundance and, as many of these play an important role in biological and chemical processes, the detection of such in vitro and in vivo is essential. Although the majority of research has focused on the development of highly selective sensors for cations and neutral molecules, more recently a large emphasis has also been placed on anion detection, and biomolecules, such as DNA, proteins, etc. as well as tissues, bones, etc. Consequently, supramolecular chemistry has become highly important in making functional and targeting molecules for use in both material sciences, where their uses include sensors, optoelectronic devices and switches, and also in biology for monitoring of complex cellular processes, such as small-molecule-messenger dynamics, enzyme activation, protein–protein interaction, receptor-protein binding, drug design and protein folding. Hence, through the major growth in the area, it is now almost possible through careful design to generate targeted synthetic molecules that can target more or less any biological molecule and function.

Together with the major developments in optoelectronics and imaging technologies in general, it is thus not surprising that such systems have been increasingly employed in the area of molecular imaging both in vitro and in vivo. This chapter gives some flavour of several examples that have been developed recently. Due to the complex nature of the field and the wide range of agents developed to date, the emphasis will be here to give examples of various types of structures, that are either supermolecules, supramolecular self-assemblies or structures that can be described as operating, due to their targeting nature, as supramolecular species. This chapter is thus not meant to be a comprehensive overview of the area, but rather an introduction to select examples of agents that have been designed and their functions within the spirit of supramolecular chemistry. Hence, this chapter is divided into two main parts: the first dealing with luminescence imaging of some sort, an area that has mainly been focused around the use of fluorescent microscopy, due to poor penetration within tissues; and agents developed for either microscopic or clinical applications. This is now a well-established area and several excellent reviews have been written on the subject in the past, which we direct interested readers toward; of particular note is a special issue in Chem. Soc. Rev. 2015 which was edited by Prof. James (University of Bath), Prof. Chang (Barkley) and the corresponding author of this contribution, Prof. Gunnlaugsson (Trinity College).¹  Other contributions on the topic have also emerged in recent times.^2–5  Hence, our aim here is to focus our discussion around a few select examples, where the emphasis is on the design and the mechanism by which these imaging agents function. Examples of agents based on naphthalimide as well as azadipyrromethene and a few other types of agents are discussed in particular, as, due to their modularity, and ease of functonalisation, these structures have become highly popular candidates for such applications.

The second part, however, focuses more on systems developed for use not only in microscopy but also for use in clinical applicants such as luminescent and magnetic resonance imaging (MRI) agents (or dual agents), the later complimenting some of the other chapters presented in this book, such as those of Bonnet and co-workers (Chapter 6) and Faulkner and co-workers (Chapter 5). We particularly focus here on the use of f-metal ion based agents, as often the choice of the metal ion employed dictates the function of the agents, such as in the case of lanthanides, where ions such as Eu(iii) and Tb(iii) are commonly employed in microscopic imaging, while Gd(iii) is employed for MRI and in clinical imaging.

In both chemical and biological systems, ions and molecules occur in abundance. Monitoring their concentrations in vitro as well as in vivo is critical as many of these ions are involved in important biological and chemical processes. For example, Na(i), K(i), Ca(ii) and Zn(ii) are involved in biological processes such as nerve impulses, muscle contraction, cell activity regulation, apoptosis and nerve transmission. The key factors for developing a luminescent agent for use in vivo are that the resulting agent has high biological stability (does not biodegrade) and has good photophysical properties (ideally, the agent should not photobleach, should have long wavelength absorption and emission properties, should have relatively long excited state lifetimes and have high quantum yields). Many such examples exist, most of them being based on the use of fluorescent systems, which emit within the visible region, with excited state lifetimes within in the ns range. Much of the literature focuses in fact on the use of classical fluorophores, which, in particular, can be photobleached within a short period of time, which can give rise to false positive or negative results. However, several examples have recently been developed where both high photostability and good or acceptable photophysical properties have been realised within a single simple organic structure. In the following sections, we present a few specific examples of those that have been developed in recent times, where the emphasis is on presenting structures that are normally highly targeted. With this in mind, we begin the discussion by examining examples where biological structures (or specific bio-targeting structures) have been incorporated into the design principle of the agent.

The term glycoconjugate is used to describe any molecule covalently linked to a carbohydrate. Glycoconjugates participate in recognition processes between cells or in the recognition of cellular structures by other molecules.⁶  These recognition events are necessary for fertilisation, cell growth and a variety of other critical cellular processes. Understanding the role of these glycoconjugates is relevant for several disciplines, such as glycobiology, molecular biology, proteomics and medicine, as changes in the glycoconjugates structures can modify their function and therefore lead to diseases.

The development of a family of glycosylated porphyrins (glycoporphyrins) and their applications for photodynamic therapy (PDT) have recently been explored by the Scanlan group.⁷  Glycoporphyrins (compounds 1a–d) containing monosaccharides, disaccharides and trisaccharides were synthesised to investigate the effects of the carbohydrate moiety on their biological behaviour, as the introduction of larger and more complex carbohydrate structures opens the possibility of specific carbohydrate–lectin interactions. The group was able to demonstrate, by employing confocal fluorescence microscopy (Figure 1.1) that the larger and partially protected saccharides improve biological compatibility as they increase water solubility, which is often an issue for therapeutic porphyrins. However, it was found that a low amount of singlet oxygen was produced and therefore the compounds exhibited low cytotoxicity toward the human oesophageal squamous carcinoma cell line.

The 1,8-naphthalimides are organic fluorophores which have been employed extensively in supramolecular chemistry.^8–10  They have visible absorbance, emit at between 500 and 650 nm and have good photostability and, hence, they have been extensively used in fluorescence sensing applications. They are also known for their therapeutic properties and, as such, are excellent candidates for use in the development of dual agents that can function both as imaging and therapeutic agents – so-called theranostic agents. Recently, they have become highly popular as imaging agents for biological applications, such as in confocal fluorescence imaging of glycosylation processes, particularly as they can be used in multiphoton excitation studies.

McCarley and co-workers^11,12  have developed naphthalimide probes for cellular imaging of tumorous environments, compounds 2 and 3. They contain a self-immolative linker (blue) and a recognition moiety (green) that selectively binds to an enzyme (quinone oxido-reductase isozyme), which is upregulated in many human cancer cells. After cellular uptake, compounds 2 and 3 are reduced by this enzyme and undergo fragmentation, thus releasing the 4-aminonaphthalimide derivatives (red), which exhibit strong fluorescence signals. Another interesting feature of using naphthalimide derivatives as fluorescent probes is their capacity to absorb two photons, opening up two-photon absorption microscopic imaging as a possibility. Two-photon microscopy (TPM) offers significant advantages because it allows for tissue penetration of up to 1 cm and therefore can be used for in vivo imaging.¹³  The near infrared window, or therapeutic window, comprises the range of wavelengths between 650 and 1350 nm, which is the range in which photons have maximal tissue penetration.¹⁴  At these wavelengths the most dominant interaction between light and tissue is scattering, which increases the distance travelled by photons within tissue, therefore increasing the absorption of photons. However, this window is narrowed by the absorption of long wavelength photons by melanin and water. TPM is based on two-photon absorption and tissue penetration is possible due to the longer excitation wavelengths used. As with regular confocal microscopy, in this process an electron is photoexcited, usually from the ground state of a molecule, to a higher energy state, such that it can then undergo relaxation to the ground via emission of light. However, rather than photoexciting the compound with a single photon of the requisite energy to populate the excited state, two photons of half the necessary energy are used instead, which, upon simultaneous absorption by the compound, allow it to populate its excited state. These lower energy photons necessarily fall within the desirable therapeutic window, making this technique attractive for in vivo purposes.

Another important feature of TPM is that it avoids using low (and hence, high energy) excitation wavelengths (lower than 405 nm) which hinder in vitro and in vivo imaging because of cell damage. Many examples have been reported in the literature exploiting this behaviour.^15,16  Yang and co-workers¹⁷  reported the first fluorescent probe for human cytochrome P450 (CYP1A) in real time using compound 4 which, upon demethylation by the CYP1A, gives the 4-hydroxy-1,8-naphthalimide 5. This hydroxyl group is deprotonated under physiological pH to give compound 6 and exhibits a broad fluorescence band upon 2-photon absorption using an excitation wavelength of 820 nm. Fan et al.¹⁸  created a fluorescence probe to monitor the level of thiols in the lysosomes of live cells using TPM. This system was also based on a ‘turn-on’ mechanism, as compound 7 possesses a sulphonamide group able to respond to thiolates, affording an ‘off–on’ signal in the lysosomes, giving compound 8, which can be imaged by TPM.

Some examples of glycosylated naphthalimides used as probes for lysosomes, intracellular thiols or senescence have been published recently.^19–21  Several groups have exploited the conjugation to a β-d-galactose unit that regulates its uptake via asialoglycoprotein receptor (ASGPR), which is overexpressed in HepG2 (a hepatic carcinoma cell line) leading to cellular internalisation.^20,22  For instance, Kim and co-workers demonstrated that only the β-d-galactose conjugated naphthalimide, compound 9, was able to undergo cellular uptake. Compound 9 possesses a self-immolative linker as the disulphide bond which is cleaved in the presence of endogenous thiols, which themselves are related to oxidative stress conditions.²³  Cleavage of the disulphide bond releases the 4-amino-1,8-naphthalimide derivative, which was detectable as it induced a redshift in the fluorescence emission and was demonstrated in vivo by using rats. In contrast, Jia and co-workers exploited the overexpression of the CD44 receptor, for hyaluronic acid, in cancer cells to deliver a naphthalimide (10) functionalised with a hyaluronic acid residue. Once inside the cells, compound 10 can be used as a cancer probe using fluorescence polarisation.²¹  Both examples rely on the overexpression of certain carbohydrate receptors to deliver the fluorescent probe inside the cells.

Recently Scanlan and co-workers have developed novel examples of fluorescent imaging agents based on the naphthalimide system, which have been conjugated to various glycans, e.g. 11 and 12. These systems were named pro-probes and were designed so that they only get delivered into biological targets upon undergoing enzymatic transformations.²⁴  Normally naphthalimides are easily taken up into cells, but by employing confocal fluorescence imaging, the authors showed that 11 and 12 were not. These were designed so that the glycosylation of the naphthalimides would serve to improve their solubility and to increase their specificity towards cancerous cells, and thus the lack of fluorescence from within the cell demonstrated that the uptake via carbohydrate-mediated receptors was not occurring. However, upon treatment with specific glycosidase enzymes the release of the naphthalimide motif occurred, via the hydrolysis of the glycosidic linkage, which resulted in the release of the naphthalimide moiety, creating not only an activatable fluorescent probe using bio-mediated release, but also achieving cell uptake only in the regions where the enzyme is upregulated (Figure 1.2). Therefore, the selective delivery of a fluorescent probe inside the cells upon enzymatic addition can be achieved and opens the possibility of using this system in vivo.

Naphthalimides have also been developed for imaging of tissues, where the release of Zn(ii) from pancreatic tissue was monitored upon administration of insulin, which is a tetramer of Zn(ii), Figure 1.3. As demonstrated by Parkesh et al., by simply attaching phenyliminodiacetate functionality to the 4-amino position of the naphthalimide structure a highly effect sensor and imaging agent, compounds 13a and b (n = 1 or 2), could be developed for Zn(ii). Compound 13a is a highly effective photoinduced electron transfer (PET) based system, which means that an active PET from the phenyliminodiacetate to the excited state of the naphthalimide unit quenches the emission, while 13b was shown to have more biological tolerance, but the PET sensing was not as affective due to the longer distance between the two components of the system. However, upon binding to Zn(ii) the PET is blocked for both (as the oxidation potential is increased) and the naphthalimide emission is switched on, and this was clear both in solution, Figure 1.3a for 13a as well as when Zn(ii) was released from pancreatic tissue that had been treated with insulin, Figure 1.3b.²⁵ 

The iminodiacetate was also shown to be an effective Ca(ii) chelator, but only at very high concentrations, and that made it be able to selectively target microcracked bone matrices, where Ca(ii) sites can be coordinated or bound to.²⁶  Examination of 13a and 13b by using epifluorescence, revealed the direct binding of both sensors to exposed Ca(ii) sites within microscratched bone, with bright green emission being observed solely within the crack against the blue emitting bone surface background (Figure 1.4). This switching ‘on’ was due to the occurrence of PET inhibition from the phenyliminodiacetate receptor to the excited state of the naphthalimide fluorophores upon successful binding of Ca(ii).

In a similar manner, the Bradley research group have greatly invested in the development of novel molecular imaging probes, recently discovering a new family of interlocked molecular architectures which showed potential for in vivo imaging of bones.²⁷  Consisting of a highly fluorescent squaraine dye encapsulated inside a protective macrocycle, these squaraine rotaxanes exhibited emission bands with deep-red wavelengths that could penetrate skin and tissue. By decorating the squaraine rotaxane core with multiple iminodiacetate groups, bone-seeking probes with dendritic molecular structures, such as 14, were generated and shown to localise to calcified areas of bone within SKH1 mice through fluorescence microscopy. Further evidence of probe 14 associating with Ca(ii) was given by the absence of fluorescent emission by the probe after pre-treatment of the bone with ethylenediaminetetraacetic acid (EDTA), a known Ca(ii) sequestering agent. Aside from histological studies, in vivo optical images revealed that 1 h after a mouse had received an injection of 14, its skeleton became strongly stained, with prominent localisation in skeletal regions of higher bone turnover, such as tibial and femoral heads, being noted. This, combined with only minor changes being seen in the skeletal fluorescence intensity over a period of 19 days, indicated great potential for quantitative long-term imaging of bone growth with probes of this nature.

Despite a number of examples in the literature reporting the iminodiacetate moiety to be a successful Ca(ii) chelator for identifying microdamage, Hyun et al. recently took an alternative approach, using sulphonate and phosphonate functionalities instead to target Ca(ii) sites within bone.²⁸  The two near infrared (NIR) fluorophores 15 and 16 were proposed as targeted contrast agents for bone, incorporating a pentamethine and heptamethine core to give rise to 700 and 800 nm fluorescence, respectively. From NIR fluorescence microscopy it was established probes 15 and 16 had a high affinity for not only the calcium salt hydroxyapatite but also for calcium phosphate. The authors suggested that whilst the phenyl sulfonates bound Ca(ii) via salt formation, the phosphonate behaved as a monodentate ligand for Ca(ii). Moreover, in vivo imaging after administration of both agents into both mice and pigs saw uptake only occur to the bones, followed by renal clearance into the urine after 24 h. Whilst microdamage was not shown to be specifically detected, the ability of such probes to selectively target hydroxyapatite or calcium phosphate within bone, through simply changing their substituents, is extremely promising.

NIR imaging also overcomes many of the problems associated with emission from biological matter, (so-called autofluorescence) which can overlap with the emission arising from the fluorescent probes, as well as penetration through biological tissue. Many examples of NIR absorbing and emitting dyes have been developed to date, such as the famous boron dipyrromethene (BODIPY) dyes and the analogue azadipyrromethene (NIR-AZA), which is also a near-infrared emitting molecule developed for biological imaging.²⁹  These probes are highly promising fluorescent structures as they also possess low energy spectral wavelengths (normally above or around 700 nm), have low toxicity and excellent photostability. O'Shea and co-workers have elegantly demonstrated their application in 2D, 3D and 4D biological imaging and have shown that, through careful design, these can be synthesised to be highly targeting imaging agents. An example of this is 17, which was developed as a fluorescent intracellular lysosome imaging agent. For its application in cellular imaging, 17 was encapsulated in 7 : 3 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) : cholesterol vesicles, that were buffered to pH 3. At this pH the emission from the probe could be easily detected at 720 nm. Using HeLa Kyoto and HeLa cell lines O'Shea, in collaboration with Gale and co-workers, demonstrated, by using real-time life imaging, that both proton and chloride transport by the transporter prodigiosin could be monitored (Figure 1.5).³⁰  The results indicated that prodigiosin can rapidly cause the pH within the lysosomal lumen to rise. This results in a non-organelle-specific increase in acidity of the cytosol which triggers cell apoptosis.³⁰ 

The design of 17 was recently modified to give 18 and 19 as bio-responsive NIR-fluorophores that could be employed as ‘switch-on’ fluorescent imaging agents for tumours. This the O'Shea group achieved in collaboration with the Elmes group by using bio-responsive cyclic arginine–glycine–aspartate (cRGD), internalizing arginine–glycine–aspartate (iRGD) and polyethylene glycol (PEG) conjugates. Live cell imaging experiments using metastatic breast cancer cells confirmed the bio-responsive capabilities in vitro and in vivo of both 18 and 19, where using the MDA-MB 231 tumour the probes ‘switched on’, and over a 96 hour period were only localised within the tumour. As had been expected, upon initial administration non-specific and very strong emission throughout was observed, and all three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. The cRGD and iRGD conjugates 18 and 19 were highly responsive and had effective tumour turn-on within the first hour of administration. The authors also demonstrated that the cRGD derivative 18 had superior specificity for tumours over the iRGD conjugate 19, Figure 1.6. The authors concluded that 18 had ‘the potential to overcome the inherent drawback of targeted always-on fluorophores requiring prolonged clearance times and shows excellent potential for clinical translation for intraoperative use in fluorescence guided tumour resections’.³¹ 

The above examples clearly demonstrate the versatility of the NIR-AZA structures in targeted biomedical imaging. This is further demonstrated with 20, which is a more simplified version of the NIR-AZA structure, exhibiting absorption and emission maxima at 686 and 716 nm, respectively and a fluorescence quantum yield of 0.18. This probe was developed, and successfully employed, as an endogenous probe for use as a NIR-fluorescent labelling agent of exosomes, without the need for immunolabelling or synthetic or chromatographic manipulation of exosomes, Figure 1.7.³² 

The above few examples demonstrate the use of fluorescence-based imaging in cells, tissues, bones and tumours. However, the problem with such agents is that often they suffer from short excited state lifetimes which can coincide with fluorescence from biological molecules. To overcome this, either long wavelength (like the example of the NIR emitting systems above by O'Shea and co-workers, which have also been employed elegantly in tumour targeting for application in laser guided surgery³³ ), or systems with long-lived excited state properties having been developed. One way of achieving the latter is to employ agents that emit from states other than the singlet excited state, as was the case for all of the above examples described so far. Notable examples of non-singlet emitters are lanthanide-based (Ln, f-metal ions) imaging agents, and their applications will be presented in the following section.

The special photophysical properties associated with the Ln(iii) ions essentially stem from the shielding of their 4f valence electrons from the chemical environment by the filled 5s² and 5p⁶ orbitals, resulting in minimal perturbation of the electronic configuration by the ligands in the first and second coordination sphere being experienced. Combining this shielding with the ability of the 4f electrons to undergo intra-configurational f–f transitions gives rise to their characteristic spectra of narrow line-like emission bands, which are ion specific, covering the entire spectrum from the ultraviolet–visible (UV–vis) to near-infrared (NIR) regions. With the exception of La(iii) and Lu(iii), the majority of the Ln(iii) ions are luminescent; while some Ln(iii) have fluorescent character, others have phosphorescent character and others display both. Photon emission which occurs via the radiative decay of spin allowed S₁ → S₀ transitions without a change in the spin conversion (ΔS = 0) is known as fluorescence, and this phenomenon generally occurs within a time frame of 10⁻¹²–10⁻⁶ s. In contrast, phosphorescence involves a change in the spin multiplicity (ΔS > 0) upon photon emission, typically via T₁ → S₀ transitions. Since it is a formally spin forbidden process, phosphorescence is generally much slower than fluorescence, ranging from 10⁻⁶ s up to several seconds.⁵  In addition to this, variations in luminescence intensity are observed across the series, with some Ln(iii) ions being more emissive than others.

The Parker research group has been involved in the development of many different lanthanide complexes for the purpose of biological probing.^34,35  One such example is the complex 21, where the Eu(iii), Tb(iii) and Yb(iii) complexes were synthesised and investigated for their potential to enantioselectively bind to human serum albumin (HSA).^36,37  In this case, the nuclear magnetic resonance (NMR) technique of saturation transfer difference (STD) was employed to investigate if these complexes bind to the protein. STD allows the detection of transient binding of small molecule ligands to macromolecular receptors, i.e. proteins and can be used to determine which part of the complex is responsible for binding to the protein, since the most strongly interacting groups of the complex will show a stronger STD effect, and requires the use of a diamagnetic ion Yb(iii). Both the SSS- and RRR-isomers were studied and it was discovered that the SSS-isomer associates selectively with the HSA compared with the RRR-enantiomer and, moreover, the drug site II of the protein was identified as directly interacting with the complex 21, principally through the phenyl methyl pendant arms and the aromatic region of the chromophore.

Complex 22 was also developed by Parker and co-workers for use in a Eu(iii) luminescence assay for the analysis of citrate in biological fluids.^38,39  Citrate detection is of importance as the levels of citrate are significantly reduced in prostate cancer tissue, and hence it can be used as a viable diagnostic marker for prostate cancer. From the results of preliminary studies it was ascertained that lactate was the main interferent in the sensing of citrate. However, the selectivity for citrate over lactate was found to be 89 : 1, which should minimise interference from variable sample lactate concentrations.

The Parker group took biological probing to a new level with the development of the Tb(iii) complex 23, which incorporated two trans-positioned azaxanthone chromophores which enabled them to visualise DNA events such as the mitotic phase, occurring in living cells, Figure 1.8.⁴⁰  The mode of action of 23 was that it would bind to the serum albumin protein in the cell, altering the coordination environment and remaining strongly emissive. Cellular uptake studies on HeLa cells using luminescence microscopy demonstrated that at relatively high concentrations (10–100 µM) the luminescent staining was consistent with endosomal and/or liposomal distribution, whereas at much lower concentrations (<1 µM), less than 10% of the observed cells were stained. It was concluded from the results of these studies that the pattern of staining, along with the type of nuclear localisation profile, was consistent with cells going through division in the ‘M’ phase of the mitotic cycle. Furthermore, a low toxicity value [concentration giving 505 of maximum inhibition (IC₅₀) > 400 µM] was estimated for complex 23. Higher resolution luminescence studies allowed the authors to follow the lifetime of a single cell stained with 23 in a cell growth medium at five-minute intervals. The evolution of the cell cycle from prophase to metaphase was clearly witnessed. However, the investigation was discontinued after an hour due to the phototoxicity associated with the intensity of the laser source used.

Compound 24 was also developed by Parker and co-workers and acts within the mitochondrial region of living cells to signal changes in bicarbonate levels.⁴¹  In fact, in-depth in vitro studies were carried out where the changes in the Ln(iii) emission of various complexes developed within the Parker group were monitored as a function of various biological analytes, including bicarbonate, HSA, citrate, lactate and phosphate. The localisation of such complexes within cells was also investigated and this study demonstrated how targeting such complexes could be performed by modulation of their pendent arm structures. A summary of this investigation is shown in Figures 1.9 and 1.10.³⁵  In the case of 24, the amide-linked azaxanthone sensitising moiety was incorporated into the Eu(iii) complex as it had been previously demonstrated to promote uptake and staining of the probe in the mitochondria of cells.³⁵  The largest changes were observed upon the addition of bicarbonate, where the ratio of the J = 1 : J = 2 band changes from 2 : 1 to >4 : 1 upon addition of the bicarbonate and displacement of the water molecules. The addition of HSA (20 µM) to 24 gave rise to a reduction in the Eu(iii) emission, in conjunction with a spectral change, with the former being attributed to the quenching of the azaxanthone triplet state by a charge transfer process from the electron rich residues in the protein. Cellular incubation studies using various cell lines, accompanied by co-localisation studies verified localisation of 24 within the mitochondrial region of the cells. The percentage of CO₂ in the incubation centre was altered and the Eu(iii) emission was found to increase with increasing pCO₂, which the authors suggested may be attributed to a rise in the steady state bicarbonate concentration.

As was mentioned above, the imaging of bone damage using non-invasive methods is of great current interest and in addition to the development of fluorescent agents for achieving such imaging, luminescent lanthanide agents have also been developed. The Eu(iii) complex 25 was the first such example, studied by McMahon et al.,⁴²  and incorporates a naphthalene antenna, which is capable of Eu(iii) sensitisation, and three iminodiacetate moieties for binding to the exposed Ca(ii) in microdamaged bone samples. Luminescent solid-state bone studies were carried out to evaluate the effectiveness of 25 as an imaging agent for microcrack damaged bone. The Eu(iii) emission spectra were recorded on scratched and smooth surfaces of the bone over various time periods, with significantly larger emission intensities being observed within the scratched region. To ensure that chelation to the Ca(ii) in the damaged bone region was occurring through the iminodiacetate moieties, analyses of the corresponding esters were also carried out, the results from which confirmed no significant interaction of the iminodiacetate functionalities with the bone's structure in their ester form. The next imaging technique employed was confocal fluorescence laser scanning microscopy. A 28-fold enhancement in the Eu(iii) emission intensity was observed in the microscratched region in comparison to the healthy bone sample. In agreement with the results of the solid-state studies, the ester analogue showed no significant contrast between damaged and healthy bone samples. A Tb(iii) version was also developed and shown to give rise to the typical green emission upon binding to the microcracks.⁴³ 

Recently, Surender et al. built on the application of luminescent lanthanide bone imaging agents by developing gold nanoparticles (AuNPs) surface functionalised with luminescent Eu(iii) complexes 26, resulting in the formation of AuNP-26·Eu·Na upon hydrolysis of the esters, and demonstrated how these particles could be used for imaging of micro-damage in bones.⁴⁴  The Eu(iii) complexes acted as calcium ion binders, as above, with strong emission elicited upon binding to Ca(ii). By using two-photon excitation (TPE) microscopy it was possible to use excitation wavelengths in the NIR region, which are desirable because of the optimal tissue penetration of NIR photons, to generate a detailed 3D map of the damaged bone (Figure 1.11). Recently the group extended this by generating both luminescent and MRI active agents, 27, and showed that by simply changing the nature of the lanthanide ion a highly versatile system could be generated, where ions such as Eu(iii) resulted in luminescent response, while Gd(iii) gave MRI responsive systems Figure 1.12, where the high population of the complexes on the AuNP 27·Gd-AuNP (upon functionalization of the AuNP with 27) give rise to excellent responses, Figure 1.13. The use of Gd(iii) in imaging will be further discussed below.

As discussed above, cellular imaging using lanthanide complexes has been elegantly developed by Parker and colleagues. More recently, Parker and co-workers have turned their focus towards developing a new class of intensely emitting Ln(iii) complexes based on the nonadentate triazacyclononane ligand framework, incorporating aryl-alkynyl groups para-substituted with either carboxylate, phenolate or phosphinate units, 28a–d, for the selective time-gated imaging of cellular mitochondria.^45,46  With each structure containing three chromophores absorbing strongly in the biologically relevant window of 310–340 nm, efficient Eu(iii) sensitisation is enabled, with such systems giving brightness an order of magnitude greater than that exhibited for the aforementioned cyclen derivatives. Defining the brightness values to be in a range of 15 000–30 000 M⁻¹ cm⁻¹, triazacyclononane Ln(iii) complexes have great potential for future roles in Förster resonance energy transfer (FRET) bio-assays and as two-photon probes. One of the first examples of these probes was 28a, which was shown by rapid spectral imaging to successfully enter the mammalian cells of the NIH-3T3, CHO and PC-3 cell lines by macropinocytosis. Subsequent trafficking to the mitochondria, which permits their selective staining, was further confirmed by colocalisation studies with the commercially available stain of Mitotracker Green.⁴⁶  The ease of structural manipulation of 28, by simply changing the functional groups incorporated into the ligand, has led to a vast array of complexes being designed, which possess different charge, hydrophilicity, pH response and intracellular localisation. In particular, alteration of the substituents on the aryl group has been identified as a method to target specific cellular organelles, staining the late endosomes and/or lysosomes, mitochondria and endoplasmic reticulum, using 28b–d, respectively.⁴⁵ 

It is clear from the above that, thanks to the long-lived excited states of Eu(iii) and Tb(iii) ions, imaging agents based on these ions have a lot to offer to the development of targeted imaging agents for use in chemical biology, as well as in medical applications. However, the latter is already well-established using lanthanide-based contrast agents for use in magnetic resonance imaging (MRI). The next section will give some overview of recent examples in this area, where the paramagnetic ion Gd(iii) has been predominantly employed.

The desire to develop responsive paramagnetic chelates that are activated by changes in physiological pH is driven by the fact that several pathologies are associated with an extracellular build-up of acid from abnormal metabolism within tissues.^47,48  By using pH to identify sites of excess acidity, particularly found in tumours, metastases and ischemic tissue, pH can be a valuable biomarker for several optical imaging methods, including the non-invasive approach of MRI. Several pH-sensitive Gd(iii) contrast agents have been reported in the literature, which were designed to induce and detect a reversible change in relaxivity in response to a variation in pH within the environment. The relaxivity of a Gd(iii) species is primarily determined by the hydration number (q), rotational correlation time (τ_R) and residence lifetimes of inner sphere water molecules (τ_M). Normally a change in one or more of these three variables is required for a pH-sensitive T₁ agent.

One of the most documented pH-sensitive MRI probes is Gd-dodecane tetraacetic acid tetraamide phosphonate (DOTA-4AmP⁵⁻), 29, which was applied in vivo in mouse kidney and rat brain glioma for extracellular tissue pH mapping using MRI. Proposed by Sherry and co-workers originally in 1999, 29 was able to respond to pH by changes in its relaxivity output upon proton exchange between the coordinated inner sphere water molecules and the surrounding bulk media.^49,50  Unlike simple tetraamide derivatives, which commonly possess high and low relaxivities in very acidic and basic media, respectively, this was not the case with 29. Instead, a gradual increase in relaxivity was noted from pH 4.5, reaching a maximum at pH 6, followed by a gentle decline until pH 8.5. This unusual pH profile of 29 as a function of relaxivity, was explained by the four appended phosphonate groups which have pK_as in the pH range 6.5–8.0 and thus hydrogen bond with the single Gd(iii) bound water molecule upon being monopronated below pH 8. This, in turn, allows for catalytic exchange of the bound water protons with those of the bulk water.⁵¹  Further development of the system by Sherry saw the amalgamation of this pH-dependent probe with a pH-independent one, which was assumed to have identical pharmacokinetics and tissue biodistributions. This meant a ‘dual-injection method’ could be utilised for estimating tissue pH by comparing the differences in MR signal intensity. Moreover, by reacting 29 with a generation five polyamidoamine (G5-PAMAM) dendrimer, a macromolecular pH sensor containing 96 molecules of the complex was successfully formed, causing a 2.2-fold increase in the relaxivity over the pH range 6.5–9. By incorporating a dendrimer, the τ_R was increased as the molecular rotation of the complex was slowed, ultimately allowing for significantly lower dosages of the complex in vivo while retaining the same changes in MRI signal as a function of pH.

Caravan is also another prominent researcher within the MRI community, who has been actively pursuing the development of contrast agents for a number of years now.⁵²  Recently, his research group designed a new pH-dependent probe 30, wherein a sulphonamide group was tethered onto the macrocyclic backbone, and they were able to achieve a dual response toward pH and HSA. Determination of the relaxivity profile of 30 in the pH region of 5.0–8.5 demonstrated the ability of the sulphonamide functionality to be protonated at low pH, with inhibition of its coordination to the Gd(iii) centre resulting in a higher relaxivity through the opening up of two sites for water ligation. Even more interesting was the pH-dependent self-aggregation of probe 30 between pH 4.5 and 5.5 where, in the absence of protein, higher relaxivity was exhibited. Introduction of HSA to the system resulted not only in an increase in the relaxivity for all the pH values of 30 but the breaking up of the aggregated species through discrete binding to the protein. Indeed, it was confirmed that only 0.5 equivalents of HSA were required to ensure complete binding of 30 to HSA, with a plateau in relaxivity being reached at 1.0 equivalents. Although the relaxivity of 30 in the presence of HSA at pH 5 was substantially lower than anticipated by the authors, due to displacement of the inner-sphere water molecules by the protein's side-chains, there is still much promise for systems of this type and other new designs as recently pointed out by Caravan and co-workers.⁵²  The use of larger self-assembly systems for such targeting is also being investigated, such as functionalising nanoparticles with targeting and responsive MRI (as well as luminescent) imaging agents.⁵³ 

Aside from these pH-responsive Gd(iii) probes, there has been a huge demand for the development of paramagnetic agents that can monitor changes in the concentration of various ions through a non-invasive method in vivo. To this end, a number of research groups have designed both alkali- and transition metal-activated MRI contrast agents in order to overcome the in vivo limitations that are associated with similar luminescent sensors, where the main drawbacks are light penetration through tissue, light scattering and toxicity caused by photobleached by-product formation.^54,55  The first example of an ion-activated MRI contrast agent was proposed by Meade and co-workers in 1999, with the q-modulated probe 31 displaying specific selectivity towards Ca(ii) ions.⁵⁶  The central motif of the probe comprised the tetraaminoacetate Ca(ii)-binding ligand 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Conjugation of this ion-binding core with two Gd(iii) cyclen-based chelates on either side, resulted in two conformational states being exhibited, the first of which entailed the acetate groups of the BAPTA ligand coordinating to the Gd(iii) centre, causing the access of solvating water to be restricted. The addition of Ca(ii) ions saw a rearrangement in the BAPTA carboxylic groups, with preferential binding to Ca(ii) over Gd(iii) being experienced and water coordination to the vacant site of the Ln(iii) ion being facilitated. Such changes in the inner coordination sphere of Gd(iii) were confirmed by an 80% increase in relaxivity in response to the presence of physiologically relevant concentrations of Ca(ii) (0.1–10 µM). More importantly, 31 showed little binding affinity for protons and Mg(ii), with only an 8% and 3% change in relaxivity, respectively, as well as remaining insensitive to pH fluctuations under physiological conditions. These key components made 31 a valuable candidate as a sensor for Ca(ii), which plays a pre-eminent role as a signalling ion in biology by functioning as an intracellular secondary messenger within the brain.

Collaboration between the groups of Logothetis and Parker has seen this MRI agent elegantly developed to produce a greater relaxivity enhancement upon the addition of Ca(ii) through modification of the central binding unit to give 32.^57,58  Incorporation of the low affinity pentadentate Ca(ii) chelator o-amino-phenol-N,N,O-triacetate (APTRA) allowed several issues to be addressed that are typically associated with high affinity chelators like BAPTA, the main one being the occurrence of binding site saturation above 1 µM concentrations of Ca(ii). In the presence of one equivalent of Ca(ii) a 97% increase in relaxivity was observed for 32, with reversibility of the Ca(ii) binding mechanism being demonstrated using EDTA. The ability of EDTA to scavenge out the Ca(ii) ions coordinated to 32 results in a decrease in the hydration number, and thus the increased relaxivity returned to its initial value of 3.5 mM⁻¹ s⁻¹. Furthermore, the applicability of 32 as an in vivo agent was verified by detecting a relaxivity response to Ca(ii) in the physiological fluids artificial cerebrospinal fluid (ACSF) and artificial extracellular matrix (AECM).

Elevated levels of Zn(ii) ions have been linked to the onset of Alzheimer's disease, whilst Zn(ii) deficiency has been connected to the progression of prostate disease and diabetes. These factors, combined with the recent awareness that Zn(ii) bound proteins play a pivotal role in controlling gene transcription and metalloenzyme function, has resulted in a number of Zn(ii)-activated probes being devised.

Matosziuk et al. reported a series of q-modulated Zn(ii) activated contrast agents, 33, by varying the length of the aliphatic alkane chain (n = 2–7) linking the Gd(iii) chelate and bioactivated Zn(ii) domain.⁵⁹  T₁ relaxivity measurements showed the greatest change in r₁ (Δr₁) in response to the binding of Zn(ii) (r_1,on) was achieved when n = 4 or 5, with a 200% increase from 2.5 to 7.8 mM⁻¹ s⁻¹ occurring. In the absence of Zn(ii) (r_1,off), the relaxivity of 33 was significantly affected by the linker length, with n = 4 or 5 giving the lowest r_1,off values. Moreover, the results of sensitivity and selectivity experiments with the metal ions Cu(ii), Mg(ii) and Ca(ii) indicated that the metal-binding interaction of 33 with Zn(ii) was independent of the distance between the Gd(iii) chelate and Zn(ii) binding motif. In the case of the hydration number the opposite was observed, with the ability of 33 to restrict water access to the Gd(iii) centre prior to the addition of Zn(ii) being strongly influenced by n. Through temperature-dependent ¹³C NMR studies the authors concluded that the linker needed to have a moderate degree of flexibility in order for the acetate groups to coordinate the Gd(iii) ion sufficiently. Intermediate linkers such as n = 4 or 5 meet this requirement, undergoing coordinative exchange with one another so that only one acetate directly binds to the Gd(iii) centre at a given time.

The Chen lab have also made great efforts in developing dual functioning MRI and fluorescent imaging probes for showing responsiveness towards Zn(ii). One of their latest examples, 34, introduced an 8-sulfonamidoquinoline sensitizer into the macrocyclic framework giving enhancements of 55% and sevenfold in both the relaxivity and fluorescence, respectively, which could be seen upon the addition of 0.5 equivalents of Zn(ii).⁶⁰  From an in vivo perspective, probe 34 demonstrated a high response for Zn(ii), with negligible changes in its relaxivity being observed in the presence of other biologically relevant cations and anions. Only in the presence of Cu(ii) was a quenching of luminescence experienced for the probe, indicating its competitive complexation to 34. Even more impressive was the ability of 34 to be harmlessly ingested and localised within the cytoplasm of breast cancer cells, displaying bright green fluorescence, which is characteristic of the quinoline moiety, under confocal microscopy. The Chen group has impressively taken this work a step further by structurally modifying the position of the amide group in 34 from the ortho to para position.⁶¹  By increasing this internal distance between the amide and 8-sulfonamidoquinoline moiety, tridentate ligand behaviour becomes inhibited. Extensive studies of this class of Zn(ii)-responsive sensors has allowed their interaction with HSA, warfarin and l-tryptophan to be better understood.

In a similar manner to Zn(ii), a number of diseases have been implicated in the misregulation of Cu(i)/Cu(ii) homeostasis, such as Wilson's and Menke's disease, prompting the development of copper-activated MRI contrast agents. The Chang lab have reported a selection of q-modulated Cu(i) agents, their most recent being probe 35, which displayed more than a 200% increase in relaxivity, with a 1 : 1 binding stoichiometry between the thioether sites of 35 and Cu(i).⁶²  Such an increment in r₁ was ascribed to the elimination of steric obstruction around the Gd(iii) centre by coordination of the pyridine nitrogen to Cu(i), thus facilitating water access. Since the reducing environment of cells favours Cu(i) over Cu(ii), the high selectivity exhibited by 35 for Cu(i) over the biologically relevant cations Na(i), K(i), Mg(ii), Ca(ii), Fe(ii), Fe(iii), Zn(ii) and Cu(ii) was extremely beneficial. Confirmation that the octaarginine tail provided efficient transport across the cell membrane was given by a tenfold increase in uptake of 35 in HEK 293T cells. Even more promising was the ability of 35 to distinguish between increases and decreases in labile Cu(i) pools of Menkes model WG1005 fibroblasts and control MCH58 fibroblasts, which were representative of diseased and healthy states, respectively. This, combined with good cell retention and low cytotoxicity, rendered the probe highly applicable for in vivo imaging of diseased cell states.

Based on the same principle of q-modulated MRI agents, Yang, Xu and co-workers published their design of a Cu(ii) responsive optical probe, 36, which induced distinct relaxivity enhancements, up to 76%, in response to the addition of 1 equivalent of Cu(ii).⁶³  This binuclear Gd(iii) chelate, with a central 2,6-bis(3-methyl-1H-pyrazol-1-yl)isonicotinic acid (BMPNA) core acting as a Cu(ii) binding scaffold, allows for the donor nitrogen of the pyrazole moiety to be selectively displaced from the Gd(iii) centre by Cu(ii), increasing the hydration number of the Ln(iii) ion and ultimately the r₁ value. Whilst the authors report that no interference was observed in this Cu(ii) triggered r₁ response towards other biologically relevant cations, the physiologically relevant anions of H₂PO₄⁻ and extracellular anion solution (EAS) did have a substantial influence on the overall relaxivity of 36, with only a maximum rise of 26% being observed. Such a reduction in r₁ was accredited to the competitive binding of these anionic species with the Ln(iii) centre, resulting in the partial displacement of the inner sphere water molecules by these anions. In addition to the above, visual changes were depicted in T₁-weighted phantom images of 36, with increases in image intensity being displayed in the presence of Cu(ii). Moreover, the clinically approved contrast agent Magnevist produced a darker image than that achieved with Yang and Xu's probe, with discernible differences also being given for Zn(ii), Mg(ii) and Ca(ii) addition.

Apart from cationic and anionic activation of MRI contrast agents, the utilisation of enzyme activation is of greater relevance, becoming a topical area of research in the past two decades. The Aime group have been long established in following gene expression in vivo through the use of MRI probes, accumulating bioactive Gd(iii) complexes within cells through the formation of oligomeric and/or polymeric structures. A β-gal gene expression MRI reporter within melanoma tumour cells was detailed by the group, consisting of a cyclen backbone bearing a galactose protected tyrosine-OH functionality, 37·Gal.⁶⁴  It was found that upon addition of the β-gal enzyme to 37·Gal, cleavage of the galactosyl-pyranose moiety occurred, with a decrease in relaxivity being witnessed for the converted lower molecular weight species 37. Successive addition of the second enzyme tyrosinase afforded an oligomerised high relaxivity system of melanin-like polymers, which was verified by a decrease in the solvent water proton relaxation time and a 2.5-fold enhancement in the nuclear magnetic relaxation dispersion (NMRD) profile. Incubation of 37·Gal with B16–F10LacZ murine melanoma cells saw a steady increase in r₁ over 30 h, further confirming the production of melanin-like macromolecules, whilst the B16–F10 cells which lacked the β-gal enzyme displayed minor changes. Moreover, by T₁-weighted MR imaging, discrimination between melanoma tumours with or without β-gal gene expression in C57Bl6 mice was achieved in the clinical range for MRI scanning.

Along with τ_R-modulated enzyme responsive probes as entailed above, several groups have also been motivated by the design of q-modulated ones, with Napolitano et al. recently presenting the first prototype of a MRI probe for glutamate decarboxylase (GAD) activity in vivo, 38.⁶⁵  The authors' design envisaged activation of 38 occurring through cleavage of the coordinated glutamate moieties upon the addition of the enzyme, which would subsequently increase the hydration state at the Gd(iii) centre. Addition of GAD to 38 indeed gave rise to a Gd(iii) complex endowed with an enhanced relaxivity, with a 60% increase noted in acidic conditions, which is the optimal pH for bacterial GAD activity. More striking was the quenching effect observed in r₁ upon the addition of chelidamic acid, a potent competitive inhibitor for GAD, which demonstrated the specificity of GAD activation for 38. Successful identification of GAD expression by T₁-weighted MR images further confirmed the high versatility of this probe for in vivo application, with future work turning to translating 38 into mammalian GAD at neutral pH.

Amphiphilic Ln(iii) complexes have a ligand structure consisting of a polar chelating terminus that is covalently linked to a hydrophobic moiety, such as a long alkyl chain or an organic molecule, which promotes their self-assembly when in aqueous solution. One of the most commonly formed aggregates by amphiphilic Ln(iii) complexes is micelles. Within these aggregates, the hydrophobic moiety is normally oriented within the interior of the cluster, whilst the hydrophilic head-group containing the Ln(iii) is exposed to the exterior environment.⁶⁶  Several Ln(iii)-based micellar systems have been reported in the literature, which focuses on understanding how the structural properties of the hydrophobic moiety influence the stability, size and aggregation number of monomers within the micelle. In particular, the length and number of the hydrophobic alkyl chains, along with the nature of the hydrophilic head-groups, have been extensively investigated.

Merbach and co-workers reported one of the first examples of monomeric Gd(iii) complexes self-organising into macromolecular assemblies, 39 and 40, by utilising the DOTA framework substituted with a monoamide-dodecyl alkyl chain for the monomers design.⁶⁷  They further modified this ligand structure to develop a series of analogous micellar systems, 41–43, which possessed different side chain lengths, varying between 10 and 18 carbon atoms.⁶⁸ 

Building upon Merbach's initial studies, Gianolio et al. developed the lipophilic Gd(iii) chelate 44, which contains a heptadentate ligand consisting of a long C₁₇ aliphatic chain covalently bound to a coordinative cage of 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid (AAZTA).⁶⁹  Possessing two coordinated water molecules (q = 2) allowed for fast water exchange to occur between 44 and the solvent and caused a threefold increase in relaxivity to be exhibited, rising from 10.2 to 30.0 s⁻¹ mM⁻¹, for the monomeric and micellar species, respectively. Replacing 98% of the Gd(iii) complexes on the external surface of the micelles with the corresponding diamagnetic Y(iii) analogue led to a further 40% enhancement in the relaxivity being observed over the entire frequency range (0.01–100 MHz). By reducing the quantity of Gd(iii) ions present within the micelles, the transverse electronic relaxation time was lengthened and the average distance between the neighbouring Gd(iii) ions increased. Furthermore, a high binding affinity for defatted HSA was displayed by 44 when in its micellar form, with a relaxivity of 84 s⁻¹ mM⁻¹ being produced, which is the highest value achieved to date. The ability of this Ln(iii) chelate to interact with one of the most abundant proteins within blood plasma, and give such a marked relaxivity enhancement, strongly emphasised its biological applicability.

The versatility of these supramolecular aggregates was further highlighted by the Desreux research group, with the click reaction being elegantly utilised to tether long aliphatic chains onto alkyne DOTA derivatives. In contrast to the aforementioned examples, the amphiphilic Gd(iii) chelate 45 featured two C₁₈ aliphatic chains directly grafted onto two adjacent carbon atoms of the tetraaza-macrocycle.⁷⁰  Monitoring the ¹H relaxation rate as a function of the Gd(iii) complex showed transition of 45 from its monomeric state to a slowly tumbling micellar species, occurred at a concentration of 4 × 10⁻³ mM. From the NMRD profile it was found the micellar aggregates had a relaxivity of 35 s⁻¹ mM⁻¹ at 20 MHz, a value six times higher than that of the parent Gd(iii) complex, which did not include the two C₁₈ alkyl moieties. Such an enhancement in r₁ was ascribed to the double anchoring of the Gd(iii) complex into the micellar structure and to the close spacing between the two octadecyl chains, both of which prevent the metal chelate and the micelle independently rotating and thus explain the high τ_R of 5206 ps and spatial restriction of the local motions, whereby S² was found to equal 0.78 (S² = 0 if the internal motion is isotropic, whilst S² = 1 if the motion is completely restricted).

The combination of dual or bimodal imaging and/or contrast agents is highly attractive, where both luminescent and MRI tags are incorporated into a single agent. Kamaly and colleagues have published several examples of bimodal contrast agents that incorporate lipophilic Gd(iii) chelates and fluorescent lipids within the bilayer membrane of liposomes.^71,72  Recently, they developed two liposomal systems capable of labelling IGROV-1 human ovarian cells in vitro and imaging xenograft tumours of mice in vivo, based on 46. Cationic and neutral liposomes with diameters of 100 nm were produced by using a formulation of either N¹-cholesteryloxycarbonyl-3-7-diazanonane-1,9-diamine (CDAN)/dioleoyl-l-α-phosphatidylethanolamine (DOPE)/46 or dioleoylphosphatidylcholine (DOPC)/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-2000)/46, in mol% ratios of 50 : 45 : 5 and 33 : 30 : 7 : 30, respectively.⁷³  These liposomal aggregates had a relaxivity of 3.6 s⁻¹ mM⁻¹ at 4.7 T, a value fourfold higher than the parent Gd(iii) complex, which lacked the fluorescent rhodamine moiety. The authors proposed this increment in r₁ was due to 46 being positioned on the outer polar surface of the bilayer membrane, which allows for the Gd(iii) chelate to be in close proximity with the surrounding bulk water. It was thought the aromatic nature of the rhodamine fluorophore prevented 46 from being deeply buried within the inner structure of the lipid bilayer. Over a sixfold increase in tumour signal enhancement, post-administration of the neutral PEGylated liposomes, was detected by MRI (T₁), with the signal strength being maintained up to 24 h. Moreover, cellular uptake and preferential localisation within tumour tissue, was verified by bright red fluorescence being observed only from the cytoplasm of carcinoma cells.

Polymeric micelles and vesicles are another type of supramolecular aggregate, which can be obtained through the self-assembly of one or more block co-polymers. Consisting of both hydrophilic and hydrophobic monomer units, these block co-polymers can exist in aqueous media as either individual molecules or lamellar structures. Typically, these amphiphilic micelle-forming monomers have a PEG block as the hydrophilic corona which shields the micelles from biologically active macromolecules. One example of polymeric micelles, published by Liu and co-workers, utilised the β-cyclodextrin (β-CD) based star copolymer 47 which contains seven Gd(iii)-DOTA complexes in the upper rim of the β-CD structure and 14poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) arms covalently conjugated with folic acid (FA) and doxorubicin (DOX), for cancer cell-targeted drug delivery and MRI contrast enhancement.⁷⁴  The authors showed that pH can be effectively used to trigger the release of the anti-cancer drug DOX from the hydrophobic core of the self-assembled nanoparticles. By simply altering the pH from 7.4 (neutral) to 4.0 (mildly acidic), the carbamate linkages between the DOX and PHPMA chains, which are acid-labile, become cleaved and consequently cause the micellar nanoparticles to disintegrate into unimer chains. Additionally, these micellar aggregates of 47 displayed a T₁ relaxivity of 11.4 s⁻¹ mM⁻¹, which was 3.7-fold larger than that of the smaller alkynyl-substituted Gd(iii)-DOTA complex. This result was further complimented by in vivo MRI studies, which saw a positive contrast enhancement in the heart, liver and kidneys of rats, for up to an hour after intravenously injecting 47 in its micellar form.

Another polymeric assembly-based theranostic system more recently reported by Liu et al. utilised the amphiphilic diblock copolymers of poly(ε-caprolactone)-b-poly(oligo(ethylene glycol)methacrylate-folic acid) [PLC-b-P(OEGMA-FA)] 48 and PLC-b-P(OEGMA-Gd) 49, covalently attached to folic acid and the Gd(iii)DOTA complex, respectively, for co-assembling mixed polymeric micelles.⁷⁵  The inner PLC hydrophobic core of these nanosized mixed micelles was loaded with the anticancer drug paclitaxel and was shown to dramatically decrease cell viability, with over 70% of HeLa cells being effectively killed by the chemotherapeutic drug being gradually released from the micelles. Besides acting as a targeted drug delivery nanocarrier, these mixed micelles also exhibited a high relaxivity of 26.29 s⁻¹ mM⁻¹, which is a value 8.3-fold higher than the r₁ of the smaller alkynyl-substituted Gd(iii)-DOTA chelate. The results of in vivo MRI studies indicated that these polymeric micelles of 48 and 49 preferentially localised within the liver of rabbits, giving over an 80% enhancement in contrast compared with the monomeric species, with signal intensity remaining constant up to 1 h post-injection.

Kim et al. have also developed cancer-recognisable MRI contrast agents that are capable of detecting small murine tumours in vivo. Polymeric micelles, with diameters of 40 nm, were self-assembled from amphiphilic block co-polymers consisting of PEG-p-l-histidine and PEG-p-l-lactide, with the latter being covalently conjugated to the Gd(iii) chelate diethylenetriaminepentaacetic acid (DTPA), 50.⁷⁶  Under physiological conditions (pH 7.4) the polymeric micelles were found to have a relaxivity of 8.56 s⁻¹ mM⁻¹ and were highly stable. However, at pH 6.5, which is the pH of tumoural environments, an increase in r₁ was exhibited, with a value of 12.01 s⁻¹ mM⁻¹ being obtained. This increment was attributed to the imidazole groups of the p(l-His) blocks being protonated at low pH, which provides the water molecules with more access to the open coordination site of the Gd(iii)–DTPA complex. Further verification of these micelles being pH sensitive was given by a 116% enhancement in the T₁ contrast being observed after 30 min and only at the site of the tumour within the mice. The authors proposed that enhanced accumulation and cellular internalisation of the contrast agent occurred as a result of the micelles breaking apart into positively charged water-soluble polymers in response to the acidic environment of the tumour.

In this chapter select examples of supramolecular imaging agents that have been developed for use in chemical biology and in the clinic are featured. Instead of giving a long list of examples, a selection of focused examples were featured which demonstrate the wide range design features and applications that such examples have to offer. Many new examples of probes and imaging agents have emerged in recent times that are not featured herein, such as the recent development in the use of aggregated induced emission (AIE) for biomolecule imaging;⁷⁷  the use of luminescent self-assembly (soft) materials and (peptide) organic particles,^78,79  or the use of coordination polymers and metal–organic frameworks (MOFs).⁸⁰  It is clear that this field is fast growing; the agents are becoming more targeted and often involving complex supramolecular engineering and self-assembly processes.

The support from Science Foundation Ireland (SFI), The Irish Research Council (IRC) and Trinity College Dublin, the University of Dublin is gratefully acknowledged.