Activatable molecular agents for cancer theranostics

Theranostics that integrates diagnosis and treatment modalities has attracted great attention due to its abilities of personalized therapy and real-time monitoring of therapeutic outcome. Such a theranostic paradigm requires agents to simultaneously possess the capabilities of targeting, imaging, and treatment. Activatable molecular agents (AMAs) are promising for cancer theranostics, as they show a higher signal-to-noise ratio (SNR), real-time detection of cancer-associated biomarkers, lower normal tissue toxicity, and a higher therapeutic effect. This perspective summarizes the recent advancements of AMAs, which include imaging-guided chemotherapy, imaging-guided photodynamic therapy, and imaging-guided photothermal therapy. The molecular design principles, theranostic mechanisms, and biomedical applications of AMAs are described, followed by a discussion of potential challenges of AMAs in cancer theranostics.

Activatable molecualr agents that intergrate diagnosis and treatment modalities have attracted great attention due to its abilities of personalized therapy and real-time monitoring of therapeutic outcome.     

The assemble,grow and lift-off (AGLO) strategy to construct complex gold nanostructures with pre-designed morphologies

The construction of metallic nanostructures with customizable morphologies and complex shapes has been an essential pursuit in nanoscience. DNA nanotechnology has enabled the fabrication of increasingly complex DNA nanostructures with unprecedented specificity, programmability and sub-nanometer precision, which makes it an ideal approach to rationally organize metallic nanostructures. Here we report an Assemble, Grow and Lift-Off (AGLO) strategy to construct robust standalone gold nanostructures with pre-designed customizable shapes in solution, using only a simple 2D DNA origami sheet as a versatile transient template. Gold nanoparticle (AuNP) seeds were firstly assembled onto the pre-designed binding sites of the DNA origami template and then additional gold was slowly deposited onto the AuNP seeds. The growing seed surfaces eventually merge with adjacent seeds to generate one continuous gold nanostructure in a pre-designed shape, which can then be lifted off the origami template. Diverse customized patterns of templated AuNP seeds were successfully transformed into corresponding gold nanostructures with the target structure transformation percentage over 80%. Moreover, the AGLO strategy can be incorporated with a magnetic bead separation platform to enable the easy recycling of the excess AuNP seeds and DNA components.

The AGLO strategy generates complex gold nanostructures with user-designed morphologies in solution, using only a simple 2D DNA origami sheet as a versatile transient template. The products are robust and stable as standalone gold nanostructures.     

The video-rate imaging of sub-10 nm plasmonic nanoparticles in a cellular medium free of background scattering

Plasmonic nanoparticles (e.g., gold, silver) have attracted much attention for biological sensing and imaging as promising nanoprobes. Practical biomedical applications demand small gold nanoparticles (Au NPs) with a comparable size to quantum dots and fluorescent proteins. Very small nanoparticles with a size below the Rayleigh limit (usually <30–40 nm) are hard to see by light scattering using a dark-field microscope, especially within a cellular medium. A photothermal microscope is able to detect very small nanoparticles, down to a few nanometers, but the imaging speed is usually too slow (minutes to hours) to image living cell processes. Here an absorption modulated scattering microscopy (AMSM) method is presented, which allows for the imaging of sub-10 nm Au NPs within a cellular medium. The unique physical mechanism of AMSM offers the remarkable ability to remove the light scattering background of the cellular component. In addition to having a sensitivity comparable to that of photothermal microscopy, AMSM has a much higher imaging speed, close to the video rate (20 fps), which allows for the dynamic tracking of small nanoparticles in living cells. This AMSM method might be a valuable tool for living cell imaging, using sub-10 nm Au NPs as biological probes, and thereby unlocking many new applications, such as single molecule labeling and the dynamic tracking of molecular interactions.

An absorption modulated scattering microscopy technique that allows for the imaging of sub-10 nm gold nanoparticles within a cellular scattering medium is presented.     

Aggregation-free and high stability core–shell polymer nanoparticles with high fullerene loading capacity,variable fullerene type,and compatibility towards biological conditions

Fullerenes have unique structural and electronic properties that make them attractive candidates for diagnostic, therapeutic, and theranostic applications. However, their poor water solubility remains a limiting factor in realizing their full biomedical potential. Here, we present an approach based on a combination of supramolecular and covalent chemistry to access well-defined fullerene-containing polymer nanoparticles with a core–shell structure. In this approach, solvophobic forces and aromatic interactions first come into play to afford a micellar structure with a poly(ethylene glycol) shell and a corannulene-based fullerene-rich core. Covalent stabilization of the supramolecular assembly then affords core-crosslinked polymer nanoparticles. The shell makes these nanoparticles biocompatible and allows them to be dried to a solid and redispersed in water without inducing interparticle aggregation. The core allows a high content of different fullerene types to be encapsulated. Finally, covalent stabilization endows nanostructures with stability against changing environmental conditions.

A polymer nanoparticle approach to biorelevant and robust fullerene nanoparticles is presented.     

Design and synthesis of a ratiometric photoacoustic imaging probe activated by selenol for visual monitoring of pathological progression of autoimmune hepatitis

Photoacoustic (PA) imaging with both the high contrast of optical imaging and the high spatial resolution of ultrasound imaging has been regarded as a robust biomedical imaging technique. Autoimmune hepatitis (AIH) is the second largest liver inflammatory disease after viral hepatitis, but its pathogenesis is not fully understood probably due to the lack of an effective in vivo monitoring approach. In this work, an innovative selenol-activated ratiometric PA imaging probe APSel was developed for visual monitoring of pathological progress of AIH. Selenols including selenocysteine (Sec, the major form of Se-containing species in vivo) have been demonstrated to have an effective antioxidant role in inflammation. The reaction of APSel with selenol results in a blue shift of the PA spectrum peak from 860 nm to 690 nm, which enables the ratiometric PA imaging. The APSel probe displays high sensitivity and selectivity to Sec and other selenols. The APSel probe was then employed for ratiometric PA imaging of selenol in cells, and for monitoring the development of AIH in a murine model by tracking the changes of selenol level. The results revealed that the level of selenol was closely correlated with the development of AIH. The proposed APSel, as the first example of a selenol-responsive PA imaging probe, provides a new tool and approach to study and diagnose AIH diseases.

A ratiometric photoacoustic imaging probe activated by selenol was developed for visual monitoring of pathological progression of autoimmune hepatitis.     

Phosphorescent metal complexes as theranostic anticancer agents: combining imaging and therapy in a single molecule

Phosphorescent metal complexes are a new kind of multifunctional antitumor compounds that can integrate imaging and antitumor functions in a single molecule. In this minireview, we summarize the recent research progress in this field, concentrating on the theranostic applications of phosphorescent iridium(iii), ruthenium(ii) and rhenium(i) complexes. The molecular design that affords these complexes with tumour- or subcellular organelle-targeting properties is elucidated. The potential of these complexes to induce and monitor the dynamic behavior of subcellular organelles and the changes in microenvironment during the process of therapy is demonstrated. Moreover, the potential and advantages of applying new technologies, such as super-resolution imaging and phosphorescence lifetime imaging, are also described. Finally, the challenges faced in the development of novel theranostic metallo-anticancer complexes for possible clinical translation are proposed.

The recent development in phosphorescent iridium, ruthenium and rhenium complexes as theranostic anticancer agents is summarized.     

A far-red fluorescent contrast agent to image epidermal growth factor receptor expression

Recent developments in optical technologies have the potential to improve the speed and accuracy of screening and diagnosis of curable precancerous lesions and early cancer, thereby decreasing the costs of detection and management of epithelial malignancies. The development of molecular-specific contrast agents for markers of early neoplastic transformation could improve the detection and molecular characterization of premalignant lesions. In the oral cavity, epidermal growth factor receptor (EGFR) overexpression has been identified in early stages of premalignant lesions of the oral squamous cell carcinoma; therefore, real-time assessment of EGFR expression could serve as a biomarker for oral neoplasia. The purpose of our study was to develop a molecular-specific optical contrast agent targeted against EGFR for in vivo assessment of epithelial neoplasia using a monoclonal antibody and the far-red fluorescent dye, Alexa Fluor 660 streptavidin. In addition to demonstrating the specificity of the contrast agent for EGFR in cell lines, we document the ability to achieve penetration through 500 microm thick epithelial layers using multilayer tissue constructs and permeability-enhancing agents. Finally, using the fluorescence intensity of the contrast agent on fresh oral cavity tissue sections, we were able to distinguish abnormal from normal oral tissue. This contrast agent should have important clinical applications for use in conjunction with fluorescence spectroscopy or imaging (or both) to facilitate tumor detection and demarcation.     

Selectively detecting attomolar concentrations of proteins using gold lined nanopores in a nanopore blockade sensor

Disease diagnosis at earlier stages requires the development of ultrasensitive biosensors for detecting low-abundance biomarkers in complex biological fluids within a reasonable time frame. Here, we demonstrate the development of an ultrasensitive nanopore blockade biosensor that can rapidly diagnose a model protein biomarker, prostate-specific antigen (PSA) with high selectivity. The solid-state nanopores have gold located only along the length of the nanopore whilst the rest of the membrane is silicon nitride. The orthogonal use of materials allows nanopore arrays with a different surface chemistry inside the nanopore relative to the rest of the membrane to be fabricated. The importance of this differential surface chemistry is it can improve the detection limit of nanopore blockade sensors in quantitative analysis. Based on such functionalized nanopore devices, nanopore blockade sensors lower the limit of detection by an order of magnitude and enable ultrasensitive detection of PSA as low as 80 aM. The findings from this study open new opportunities for nanopore sensors in further developments including optical detection and ultralow detection limit biosensing at complex biological fluids.

Selective detection of attomolar proteins was achieved using gold lined nanopores in a nanopore blockade sensor.     

Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging

Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies on optical imaging, yet these membranous hubs of bioenergy are exceptionally vulnerable to photodamage. We report that cyclooctatetraene-conjugated cyanine dyes (PK Mito dyes), are ideal mitochondrial probes with remarkably low photodynamic damage for general use in fluorescence cytometry. In contrast, the nitrobenzene conjugate of Cy3 exhibits enhanced photostability but unaffected phototoxicity compared to parental Cy3. PK Mito Red, in conjunction with Hessian-structural illumination microscopy, enables 2000-frame time-lapse imaging with clearly resolvable crista structures, revealing rich mitochondrial dynamics. In a rigorous stem cell sorting and transplantation assay, PK Mito Red maximally retains the stemness of planarian neoblasts, exhibiting excellent multifaceted biocompatibility. Resonating with the ongoing theme of reducing photodamage using optical approaches, this work advocates the evaluation and minimization of phototoxicity when developing imaging probes.

Cyclooctatetraene-conjugated cyanine dyes represent an effective strategy to improve biocompatibility under light in live-cell fluorescence imaging and analysis of mitochondria.     

Nanoshell magnetic resonance imaging contrast agents

Nanocontrast agents have great potential in magnetic resonance (MR) molecular imaging applications for clinical diagnosis. We synthesized Au(3)Cu(1) (gold and copper) nanoshells that showed a promising MR contrast effect. For in vitro MR images, the large proton r1 relaxivities brightened T(1)-weighted images. As for the proton-dephasing effect in T(2), Au(3)Cu(1) lightened MR images at the low concentration of 0.125 mg mL(-1) (3.84 x 10(-7) mM), and then the signal continuously decreased as the concentration increased. For in vivo MR imaging, Au(3)Cu(1) nanocontrast agents enhanced the contrast of blood vessels and suggested their potential use in MR angiography as blood-pool agents. We propose that (1) the cooperativity originating from the form of the nanoparticles and (2) the large surface area coordinated to water from their porous hollow morphology are important for efficient relaxivity. In a cytotoxicity and animal survival assay, Au(3)Cu(1) nanocontrast agents showed a dose-dependent toxic effect: the viability rate of experimental mice reached 83% at a dose of 20 mg kg(-1) and as much as 100% at 2 mg kg(-1).     

Metallohelices emulate the properties of short cationic α-helical peptides

Naturally occurring peptides in many living systems perform antimicrobial and anticancer host defence roles, but their potential for clinical application is limited by low metabolic stability and relatively high costs of goods. Self-assembled helical metal complexes provide an attractive synthetic platform for non-peptidic architectures that can emulate some of the properties of short cationic α-helical peptides, with tuneable charge, shape, size and amphipathicity. Correspondingly there is a growing body of evidence demonstrating that these supramolecular architectures exhibit bioactivity that emulates that of the natural systems. We review that evidence in the context of synthetic advances in the area, driven by the potential for biomedical applications. We note some design considerations for new biologically-relevant metallohelices, and give our outlook on the future of these compounds as therapeutic peptidomimetics.

Advances in metal–organic self-assembly have unearthed an area of chemical space containing peptide-emulating molecules. Comparison with natural systems indicates future directions in terms of synthesis, disease areas and mechanistic understanding.     

Palladium: a future key player in the nanomedical field?

Metal nanostructures offer invaluable possibilities for targeted drug delivery, detection/diagnosis and imaging. Whereas iron, gold, silver and platinum nanoarchitectures have largely dominated this field to date, several hurdles impede the widespread application of those nanopharmaceuticals in a clinical context. Therefore, technologies based on alternative metals are now being evaluated for their potential in medical applications. Palladium nanostructures are characterized by remarkable catalytic and optical properties. However, until recently, very few studies have taken advantage of these unique characteristics for applications in the biomedical field. Very recently, palladium nanostructures have been reported as prodrug activator, as photothermal agents and for anti-cancer/anti-microbial therapy. With only a handful of reports available, the pharmaceutical applications of palladium nanostructures reviewed here are in their infancy. Yet their interesting performance and toxicity profiles may qualify them as future key players in the nanomedical field.     

SERS detection of streptavidin/biotin monolayer assemblies

A two-dimensional array of gold nanotriangles inscribed onto glass coverslips were optimized for the surface-enhanced Raman detection of streptavidin/biotin monolayer assemblies. The nanostructures were fabricated by electron beam lithography, and its optical parameters were optimized to be probed under a Raman microscope with a linearly polarized He-Ne laser with an excitation wavelength of λ = 632.8 nm. The platforms were first tested against a monolayer of biotinylated alkanethiols (BAT) functionalized over the gold nanostructure, showing that good-quality spectra could be acquired with a short acquisition time. The supramolecular interaction of streptavidin (strep) with BAT showed subsequent modification of the Raman spectrum that implies a change in the secondary structure of the host biomolecule (streptavidin). Compared to gold surfaces without nanoscale structures, the local enhancement that results from our nanostructured surfaces allows one to detect the vibrational signal of monolayers within a time on the order of seconds and under modest laser intensity, further demonstrating the utility of using plasmonic metallic nanostructures for molecular recognition.     

Synthesis of an AIEgen functionalized cucurbit[7]uril for subcellular bioimaging and synergistic photodynamic therapy and supramolecular chemotherapy

Aggregation-induced emission (AIE) based fluorophores (AIEgens) have attracted increasing attention for biomedical applications due to their unique optical properties. Here we report an AIE photosensitizer functionalized CB[7], namely AIECB[7], which could spontaneously self-assemble into nanoaggregates in aqueous solutions. Interestingly, the carbonyl-lace of CB[7] may potentially act as a proton acceptor in an acidic environment to fine-tune the fluorescence and singlet oxygen generation of AIECB[7] nanoaggregates by regulating the inner stacking of AIEgens. Additionally, benefiting from the guest-binding properties of CB[7], oxaliplatin was included into AIECB[7] nanoaggregates for combined photodynamic therapy and supramolecular chemotherapy. To show the modular versatility of this supramolecular system, a hypoxia-activatable prodrug banoxantrone (AQ4N) was loaded into AIECB[7] nanoaggregates, which exhibited synergistic antitumor effects on a multicellular tumor spheroid model (MCTS). This work not only provides AIECB[7] for versatile theranostic applications, but also offers important new insights into the design and development of macrocycle-conjugated AIE materials for diverse biomedical applications.

An AIEgen-functionalized cucurbit[7]uril was synthesized for the first time and spontaneously self-assembled into nanoaggregates in aqueous solutions and allowed subcellular imaging of the lysosome and photodynamic therapy and chemotherapy of cancer.     

Hydrophilic cyanine dyes as contrast agents for near-infrared tumor imaging: synthesis, photophysical properties and spectroscopic in vivo characterization

We have synthesized a group of glucamine and gluosamine-substituted cyanine dyes structurally related to indocyanine green (ICG) and have characterized these compounds with regard to their potential as contrast agents for biomedical optical imaging. The compounds reported herein exhibit increased hydrophilicity and less plasma protein binding (< 50%), and are thus expected to have different pharmacokinetic properties compared with ICG. Furthermore, we measured enhanced fluorescence quantum yields (7-15%) in a physiological environment with respect to ICG. For the derivative with the highest hydrophilicity (5a) the efflux from tumor and normal tissue was monitored by intensity-modulated diffuse optical spectroscopy after intravenous injection into tumor-bearing rats. In comparison with ICG, 5a exhibited a considerably enhanced tissue-efflux half-life (73 min versus less than 10 min for ICG in tumor tissue), a two-fold higher initial tissue absorption coefficient compared to ICG, and finally, it generated an elevated tumor-to-tissue concentration gradient up to 1 h after injection. In conclusion, compounds such as 5a are promising contrast agents for optical imaging, and could facilitate highly sensitive and specific detection of breast cancer or other malignancies by utilizing mechanisms similar to contrast-enhanced magnetic resonance imaging or computerized tomography.     

Recent advances of AIE light-up probes for photodynamic therapy

As a new non-invasive treatment method, photodynamic therapy (PDT) has attracted great attention in biomedical applications. The advantages of possessing fluorescence for photosensitizers have made it possible to combine imaging and diagnosis together with PDT. The unique features of aggregation-induced emission (AIE) fluorogens provide new opportunities for facile design of light-up probes with high signal-to-noise ratios and improved theranostic accuracy and efficacy for image-guided PDT. In this review, we summarize the recent advances of AIE light-up probes for PDT. The strategies and principles to design AIE photosensitizers and light-up probes are firstly introduced. The application of AIE light-up probes in photodynamic antitumor and antibacterial applications is further elaborated in detail, from binding/targeting-mediated, reaction-mediated, and external stimuli-mediated light-up aspects. The challenges and future perspectives of AIE light-up probes in the PDT field are also presented with the hope to encourage more promising developments of AIE materials for phototheranostic applications and translational research.

AIE fluorogens provide new opportunities for the development of light-up probes for photodynamic therapy.     

Polymerizable Gd(iii) building blocks for the synthesis of high relaxivity macromolecular MRI contrast agents

A new synthetic strategy for the preparation of macromolecular MRI contrast agents (CAs) is reported. Four gadolinium(iii) complexes bearing either one or two polymerizable methacrylamide groups were synthesized, serving as monomers or crosslinkers for the preparation of water-soluble, polymeric CAs using Reversible Addition–Fragmentation Chain Transfer (RAFT) polymerization. Using this approach, macromolecular CAs were synthesized with different architectures, including linear, hyperbranched polymers and gels. The relaxivities of the polymeric CAs were determined by NMR relaxometry, revealing an up to 5-fold increase in relaxivity (60 MHz, 310 K) for the linear polymers compared with the clinically used CA, Gd-DOTA. Moreover, hyperbranched polymers obtained from Gd(iii) crosslinkers, displayed even higher relaxivities up to 22.8 mM⁻¹ s⁻¹, approximately 8 times higher than that of Gd-DOTA (60 MHz, 310 K). A detailed NMRD study revealed that the enhanced relaxivities of the hyperbranched polymers were obtained by limiting the local motion of the crosslinked Gd(iii) chelate. The versatility of RAFT polymerization of Gd(iii) monomers and crosslinkers opens the doors to more advanced polymeric CAs capable of multimodal, bioresponsive or targeting properties.

A new synthetic strategy for the preparation of efficient macromolecular MRI contrast agents is reported.     

Optical imaging with dynamic contrast agents

Biological imaging applications often employ molecular probes or nanoparticles for enhanced contrast. However, resolution and detection are still often limited by the intrinsic heterogeneity of the sample, which can produce high levels of background that obscure the signals of interest. Herein, we describe approaches to overcome this obstacle based on the concept of dynamic contrast: a strategy for elucidating signals by the suppression or removal of background noise. Dynamic contrast mechanisms can greatly reduce the loading requirement of contrast agents, and may be especially useful for single-probe imaging. Dynamic contrast modalities are also platform-independent, and can enhance the performance of sophisticated biomedical imaging systems or simple optical microscopes alike. Dynamic contrast is performed in two stages: 1) a signal modulation scheme to introduce time-dependent changes in amplitude or phase, and 2) a demodulation step for signal recovery. Optical signals can be coupled with magnetic nanoparticles, photoswitchable probes, or plasmon-resonant nanostructures for modulation by magnetomotive, photonic, or photothermal mechanisms, respectively. With respect to image demodulation, many of the strategies developed for signal processing in electronics and communication technologies can also be applied toward the editing of digital images. The image-processing step can be as simple as differential imaging, or may involve multiple reference points for deconvolution by using cross-correlation algorithms. Periodic signals are particularly amenable to image demodulation strategies based on Fourier transform; the contrast of the demodulated signal increases with acquisition time, and modulation frequencies in the kHz range are possible. Dynamic contrast is an emerging topic with considerable room for development, both with respect to molecular or nanoscale probes for signal modulation, and also to methods for more efficient image processing and editing.     

Mapping blood biochemistry by Raman spectroscopy at the cellular level

We report how Raman difference imaging provides insight on cellular biochemistry in vivo as a function of sub-cellular dimensions and the cellular environment. We show that this approach offers a sensitive diagnostic to address blood biochemistry at the cellular level. We examine Raman microscopic images of the distribution of the different hemoglobins in both healthy (discocyte) and unhealthy (echinocyte) blood cells and interpret these images using pre-calculated, accurate pre-resonant Raman tensors for scattering intensities specific to hemoglobins. These tensors are developed from theoretical calculations of models of the oxy, deoxy and met forms of heme benchmarked against the experimental visible spectra of the corresponding hemoglobins. The calculations also enable assignments of the electronic transitions responsible for the colour of blood: these are mainly ligand to metal charge transfer transitions.

We assign the electronic transitions responsible for the colour of blood and present a Raman imaging diagnostic approach for individual blood cells.