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Design of Water-Soluble Rotaxane-Capped Superparamagnetic, Ultrasmall Fe3O4 Nanoparticles for Targeted NIR Fluorescence Imaging in Combination with Magnetic Resonance Imaging
Abstract
Integrating an NIR fluorescent probe with a magnetic resonance imaging (MRI) agent to harvest complementary imaging information is challenging. Here, we have designed water-soluble, biocompatible, noncytotoxic, bright-NIR-emitting, sugar-functionalized, mechanically interlocked molecules (MIMs)-capped superparamagnetic ultrasmall Fe3O4 NPs for targeted multimodal imaging. Dual-functional stoppers containing an unsymmetrical NIR squaraine dye interlocked within a macrocycle to construct multifunctional MIMs are developed with enhanced NIR fluorescence efficiency and durability. One of the stoppers of the axle is composed of a lipophilic cationic TPP+ functionality to target mitochondria, and the other stopper comprises a dopamine-containing catechol group to anchor at the surface of the synthesized Fe3O4 NPs. Fe3O4 NPs surface-coated with targeted NIR rotaxanes help to deliver ultrasmall magnetic NPs specifically inside the mitochondria. Two carbohydrate moieties are conjugated with the macrocycle of the rotaxane via click chemistry to improve the water solubility of MitoSQRot-(Carb-OH)2-DOPA-Fe3O4 NPs. Water-soluble, rotaxane-capped Fe3O4 NPs are used for live-cell mitochondria-targeted NIR fluorescence confocal imaging, 3D and multicolor imaging in combination with T2-weighted MRI on a 9.4 T MR scanner with a high relaxation rate (r2) of 180.7 mM-1 s-1. Biocompatible, noncytotoxic, ultrabright NIR rotaxane-capped superparamagnetic ultrasmall monodisperse Fe3O4 NPs could be a promising agent for targeted multimodal imaging applications.
... Therefore, alongside improving the treatment of primary lesions, monitoring cancer metastasis is crucial [27,28]. In recent years, various NPs with diagnostic capabilities have emerged to detect metastatic cancers using techniques such as fluorescence imaging, magnetic resonance imaging (MRI), photoacoustic imaging and et al. [29][30][31]. The combined application of multiple imaging modalities not only enhances diagnostic accuracy and cancer localization but also effectively tracks and monitors cancer progression in real time, contributing to the development of more personalized and effective treatment plans [32][33][34]. ...
To address the limitations of traditional photothermal therapy (PTT)/ photodynamic therapy (PDT) and real-time cancer metastasis detection, a pH-responsive nanoplatform (NP) with dual-modality imaging capability was rationally designed. Herein, 1 H,1 H-undecafluorohexylamine (PFC), served as both an oxygen carrier and a ¹⁹F magnetic resonance imaging (MRI) probe, and photosensitizer indocyanine green (ICG) were grafted onto the pH-responsive peptide hexahistidine (H6) to form H6-PFC-ICG (HPI). Subsequently, the heat shock protein 90 inhibitor, gambogic acid (GA), was incorporated into hyaluronic acid (HA) modified HPI (HHPI), yielding the ultimate HHPI@GA NPs. Upon self-assembly, HHPI@GA NPs passively accumulated in tumor tissues, facilitating oxygen release and HA-mediated cell uptake. Once phagocytosed by lysosomes, protonation of H6 was triggered due to the low pH, resulting in the release of GA. With near-infrared laser irradiation, GA-mediated decreased HSP90 expression and PFC-mediated increased ROS generation amplified the PTT/PDT effect of HHPI@GA, leading to excellent in vitro and in vivo anticancer efficacies. Additionally, the fluorescence and ¹⁹F MRI dual-imaging capabilities of HHPI@GA NPs enabled effective real-time primary cancer and lung metastasis monitoring. This work offers a novel approach for enhanced cancer phototherapy, as well as precise cancer diagnosis.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12951-024-02431-6.
... In addition, the probe has strong red fluorescence, which has the advantages of eliminating the influence of cell fluorescence, less dispersion, and increased tissue penetration. 34 Therefore, Met-NHs-AuNCs has a promising application in targeted tumor imaging. ...
Background
The preclinical diagnosis of tumors is of great significance to cancer treatment. Near-infrared fluorescence imaging technology is promising for the in-situ detection of tumors with high sensitivity.
Methods
Here, a fluorescent probe was synthesized on the basis of Au nanoclusters with near-infrared light emission and applied to fluorescent cancer cell labeling. Near-infrared methionine-N-Hydroxy succinimide Au nanoclusters (Met-NHs-AuNCs) were prepared successfully by one-pot synthesis using Au nanoclusters, methionine, and N-Hydroxy succinimide as frameworks, reductants, and stabilizers, respectively. The specific fluorescence imaging of tumor cells or tissues by fluorescent probe was studied on the basis of SYBYL Surflex-DOCK simulation model of LAT1 active site of overexpressed receptor on cancer cell surface. The results showed that Met-NHs-AuNCs interacted with the surface of LAT1, and C_Score scored the conformation of the probe and LAT1 as five.
Results
Characterization and in vitro experiments were conducted to explore the Met-NHs-AuNCs targeted uptake of cancer cells. The prepared near-infrared fluorescent probe (Met-NHs-AuNCs) can specifically recognize the overexpression of L-type amino acid transporter 1 (LAT1) in cancer cells so that it can show red fluorescence in cancer cells. Meanwhile, normal cells (H9c2) have no fluorescence.
Conclusion
The fluorescent probe demonstrates the power of targeting and imaging cancer cells.
Multimodal imaging, which combines the strengths of two or more imaging modalities to provide complementary anatomical and molecular information, has emerged as a robust technology for enhancing diagnostic sensitivity and accuracy, as well as improving treatment monitoring. Moreover, the application of multimodal imaging in guiding precision tumor treatment can prevent under- or over-treatment, thereby maximizing the benefits for tumor patients. In recent years, several intriguing magneto-optical nanosystems with both magnetic and optical properties have been developed, leading to significant breakthroughs in the field of multimodal imaging and image-guided tumor therapy. These advancements pave the way for precise tumor medicine. This review summarizes various types of magneto-optical nanosystems developed recently and describes their applications as probes for multimodal imaging and agents for image-guided therapeutic interventions. Finally, future research and development prospects of magneto-optical nanosystems are discussed along with an outlook on their further applications in the biomedical field.
Research into mechanically interlocked luminescent molecules (MILMs), which is the overlapping of mechanically interlocked molecules and luminescent molecules, has intensified over the past few decades. These studies have tapped into and exploited the benefits of mechanically interlocked structures to achieve outstanding and stimulus‐responsive optical characteristics, resulting in the synthesis of new types of luminescent systems and exploring their potential uses in different applications. This review describes the salient attributes of MILMs and showcases some of the latest advancements in this field of research.
Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800‐1 have been tested in humans. However, long‐term imaging protocols using ZW800‐1 conjugates are limited by their instability, primarily because the chemically labile C4′‐O‐aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800‐1, with greatly enhanced dye stability. NIR‐I and NIR‐II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor‐targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.
Multimodal imaging, which harnesses two or more imaging modalities to produce complementary anatomical and molecular information of a living subject, has become as a powerful tool in both basic biomedical research and clinical diagnosis. The progresses in multimodal imaging are paralleled by the advances in multimodal probes, particularly activatable multimodal imaging probes that can generate concurrent switches in different imaging modality signals upon interaction with a molecular target. These probes are extremely promising for in vivo imaging. In this Minireview, we summarize the recent progress in activatable multimodal probes for in vivo imaging and cancer theranostics, focusing on their design principle, signal activation mechanism and biomedical applications. The current challenges and perspectives for future developments of activatable multimodal probes are also briefly discussed. We hope that this Minireview will provide inspiration for the design of other activatable multimodal probes for improving in vivo imaging and theranostics.
The arrival of cell-based therapies is a revolution in medicine. However, its safe clinical application in a rational manner depends on reliable, clinically applicable methods for determining the fate and trafficking of therapeutic cells in vivo using medical imaging techniques─known as in vivo cell tracking. Radionuclide imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) has several advantages over other imaging modalities for cell tracking because of its high sensitivity (requiring low amounts of probe per cell for imaging) and whole-body quantitative imaging capability using clinically available scanners. For cell tracking with radionuclides, ex vivo direct cell radiolabeling, that is, radiolabeling cells before their administration, is the simplest and most robust method, allowing labeling of any cell type without the need for genetic modification. This Review covers the development and application of direct cell radiolabeling probes utilizing a variety of chemical approaches: organic and inorganic/coordination (radio)chemistry, nanomaterials, and biochemistry. We describe the key early developments and the most recent advances in the field, identifying advantages and disadvantages of the different approaches and informing future development and choice of methods for clinical and preclinical application.
Mechanically interlocked molecules present opportunities to construct therapeutic drugs and diagnostic imaging agents but harnessing supramolecular chemistry to make biologically active probes in water is a challenge. Here, we describe a rotaxane‐based approach to synthesise radiolabelled proteins and peptides for molecular imaging of cancer biomarkers in vivo. Host–guest chemistry using β‐cyclodextrin‐ and cucurbit[6]uril‐catalysed cooperative capture synthesis produced gallium‐68 or zirconium‐89 radiolabelled metallo[4]rotaxanes. Photochemical conjugation to trastuzumab led to a viable positron emission tomography (PET) radiotracer. The rotaxane architecture can be tuned to accommodate different radiometal ion complexes, other protein‐ or peptide‐based drugs, and fluorophores for optical detection. This technology provides a platform to explore how mechanical bonding can improve drug delivery, enhance tumour specificity, control radiotracer pharmacokinetics, and reduce dosimetry.
A general synthetic method creates a new class of covalently connected, self‐threaded, fluorescent molecular probes with figure‐eight topology, an encapsulated deep‐red fluorophore, and two peripheral peptide loops. The globular molecular shape and rigidified peptide loops enhance imaging performance by promoting water solubility, eliminating probe self‐aggregation, and increasing probe stability. Moreover, the peptide loops determine the affinity and selectivity for targets within complex biological samples such as cell culture, tissue histology slices, or living subjects. For example, a probe with cell‐penetrating peptide loops targets the surface of cell plasma membranes, whereas, a probe with bone‐targeting peptide loops selectively stains the skeleton within a living mouse. The unique combination of bright deep‐red fluorescence, high stability, and predictable peptide‐based targeting is ideal for photon intense fluorescence microscopy and biological imaging.
The second near-infrared wavelength window (NIR-II, 1,000–1,700 nm) enables fluorescence imaging of tissue with enhanced contrast at depths of millimetres and at micrometre-scale resolution. However, the lack of clinically viable NIR-II equipment has hindered the clinical translation of NIR-II imaging. Here, we describe an optical-imaging instrument that integrates a visible multispectral imaging system with the detection of NIR-II and NIR-I (700–900 nm in wavelength) fluorescence (by using the dye indocyanine green) for aiding the fluorescence-guided surgical resection of primary and metastatic liver tumours in 23 patients. We found that, compared with NIR-I imaging, intraoperative NIR-II imaging provided a higher tumour-detection sensitivity (100% versus 90.6%; with 95% confidence intervals of 89.1%–100% and 75.0%–98.0%, respectively), a higher tumour-to-normal-liver-tissue signal ratio (5.33 versus 1.45) and an enhanced tumour-detection rate (56.41% versus 46.15%). We infer that combining the NIR-I/II spectral windows and suitable fluorescence probes might improve image-guided surgery in the clinic. An optical-imaging instrument that integrates a visible multispectral imaging system with the detection of near-infrared fluorescence in the first and second windows aids the fluorescence-guided surgical resection of liver tumours in patients.
Surgical resection is the primary and most effective treatment for most patients with solid tumors. However, patients suffer from postoperative recurrence and metastasis. In the past years, emerging nanotechnology has led the way to minimally invasive, precision and intelligent oncological surgery after the rapid development of minimally invasive surgical technology. Advanced nanotechnology in the construction of nanomaterials (NMs) for precision imaging‐guided surgery (IGS) as well as surgery‐assisted synergistic therapy is summarized, thereby unlocking the advantages of nanotechnology in multimodal IGS‐assisted precision synergistic cancer therapy. First, mechanisms and principles of NMs to surgical targets are briefly introduced. Multimodal imaging based on molecular imaging technologies provides a practical method to achieve intraoperative visualization with high resolution and deep tissue penetration. Moreover, multifunctional NMs synergize surgery with adjuvant therapy (e.g., chemotherapy, immunotherapy, phototherapy) to eliminate residual lesions. Finally, key issues in the development of ideal theranostic NMs associated with surgical applications and challenges of clinical transformation are discussed to push forward further development of NMs for multimodal IGS‐assisted precision synergistic cancer therapy. State‐of‐the‐art advances in advanced nanotechnology for precision surgery are presented, with emphasis on multimodal imaging‐guided precision surgery for intraoperative visualization and synergistic surgical therapy for the elimination of residual lesions. These are of great value to push forward the development of nanomaterials in oncological surgery and clinical translation.
We report a novel assembly of polyethyleneimine (PEI)-coated Fe3O4 nanoparticles (NPs) with single-stranded DNA (ssDNA), and the fluorescence of the dye labeled in the DNA is remarkably quenched. In the presence of a target protein, the protein–DNA aptamer mutual interaction releases the ssDNA from this assembly and hence restores the fluorescence. This feature could be adopted to develop an aptasensor for protein detection. As a proof-of-concept, for the first time, we have used this proposed sensing strategy to detect thrombin selectively and sensitively. Furthermore, simultaneous multiple detection of thrombin and lysozyme in a complex protein mixture has been proven to be possible.
Magnetic resonance imaging (MRI) is a highly valuable non-invasive imaging tool owing to its exquisite soft tissue contrast, high spatial resolution, lack of ionizing radiation, and wide clinical applicability. Contrast agents (CAs) can be used to further enhance the sensitivity of MRI to obtain information-rich images. Recently, extensive research efforts have been focused on the design and synthesis of high-performance inorganic nanoparticle-based CAs to improve the quality and specificity of MRI. Herein, the basic rules, including the choice of metal ions, effect of electron motion on water relaxation, and involved mechanisms, of CAs for MRI have been elucidated in detail. In particular, various design principles, including size control, surface modification (e.g. organic ligand, silica shell, and inorganic nanolayers), and shape regulation, to impact relaxation of water molecules have been discussed in detail. Comprehensive understanding of how these factors work can guide the engineering of future inorganic nanoparticles with high relaxivity. Finally, we have summarized the currently available strategies and their mechanism for obtaining high-performance CAs and discussed the challenges and future developments of nanoparticulate CAs for clinical translation in MRI.
Significance
The main goal in the emerging field of cancer nanomedicine is to generate, standardize, and produce multifunctional carriers designed to improve the response of drugs against tumors. Here we report the design, development, and preclinical validation of a ligand-directed bioinorganic platform that integrates tumor targeting, receptor-mediated cell internalization, photon-to-heat conversion, and drug delivery. This enabling hydrogel-based technology can accommodate a broad variety of ligands, nanoparticles, and payloads. We show experimental proof-of-concept in mouse models of breast and prostate cancer with molecular imaging and marked reduction of tumor growth. However, with future proof that this technology is translatable, medical applications beyond cancer may also be leveraged.
An efficient magnetic resonance imaging (MRI) contrast agent with high R2 relaxivity value is achieved by controlling the shape of iron oxide to a rod like morphology with a length 30-70 nm and diameter 4-12 nm. Fe3O4 nanorods of length 70 nm, encapsulated with polyethyleneimine show very high R2 relaxivity value of 608 mM−1s−1. The enhanced MRI contrast of nanorods is attributed to higher surface area and anisotropic morphology. The higher surface area induces a stronger magnetic field perturbation over a larger volume more effectively for the outer sphere protons. The shape anisotropy contribution is understood by calculating the local magnetic field of nanorod and nanoparticle under an applied magnetic field (3 Tesla). As compared to spherical geometry, the induced magnetic field of rod is stronger and hence the strong magnetic field over a large volume leads to higher R2 relaxivity for nanorods.
We describe the design, synthesis and in vitro evaluation of a multimodal and multimeric contrast agent. The agent consists of three macrocyclic Gd(iii) chelates conjugated to a fluorophore and possesses high relaxivity, water solubility, and is nontoxic. The modular synthesis is amenable for the incorporation of a variety of fluorophores to generate molecular constructs for a number of applications.
Superparamagnetic iron oxide (SPIO) MR contrast agents are composed of nano-sized iron oxide crystals coated with dextran or carboxydextran. Two SPIO agents are clinically approved, namely: ferumoxides (Feridex in the USA, Endorem in Europe) with a particle size of 120 to 180 nm, and ferucarbotran (Resovist) with a particle size of about 60 nm. The principal effect of the SPIO particles is on T2* relaxation and thus MR imaging is usually performed using T2/T2*-weighted sequences in which the tissue signal loss is due to the susceptibility effects of the iron oxide core. Enhancement on T1-weighted images can also be seen with the smaller Resovist. Both Feridex and Resovist are approved specifically for MRI of the liver. The difference being that Resovist can be administered as a rapid bolus (and thus can be used with both dynamic and delayed imaging), whereas Feridex needs to be administered as a slow infusion and is used solely in delayed phase imaging. In the liver, these particles are sequestered by phagocytic Kupffer cells in normal reticuloendothelial system (RES), but are not retained in lesions lacking Kupffer cells. Consequently, there are significant differences in T2/T2* relaxation between normal tissue and lesions, resulting in increased lesion conspicuity and detectability. SPIO substantially increase the detectability of hepatic metastases. For focal hepatocellular lesions, SPIO-enhanced MR imaging exhibits slightly better diagnostic performance than dynamic CT. A combination of dynamic and static MR imaging technique using T1- and T2 imaging criteria appears to provide clinically more useful patterns of enhancement. Feridex and Resovist are also used for evaluating macrophage activities in some inflammatory lesions, but their clinical values remain to be further confirmed. The clinical development of Ferumoxtran (Combidex in the USA, Sinerem in Europe), designed for lymph node metastasis evaluation, is currently stopped.
A new gadolinium chelating NIR fluorescent molecular probe increases T(1) relaxivity of water protons, facilitating combined optical and magnetic resonance imaging.
The development of nanocrystals has been intensively pursued, not only for their fundamental scientific interest, but also for many technological applications. The synthesis of monodisperse nanocrystals (size variation <5%) is of key importance, because the properties of these nanocrystals depend strongly on their dimensions. For example, the colour sharpness of semiconductor nanocrystal-based optical devices is strongly dependent on the uniformity of the nanocrystals, and monodisperse magnetic nanocrystals are critical for the next-generation multi-terabit magnetic storage media. For these monodisperse nanocrystals to be used, an economical mass-production method needs to be developed. Unfortunately, however, in most syntheses reported so far, only sub-gram quantities of monodisperse nanocrystals were produced. Uniform-sized nanocrystals of CdSe (refs 10,11) and Au (refs 12,13) have been produced using colloidal chemical synthetic procedures. In addition, monodisperse magnetic nanocrystals such as Fe (refs 14,15), Co (refs 16-18), gamma-Fe(2)O(3) (refs 19,20), and Fe(3)O(4) (refs 21,22) have been synthesized by using various synthetic methods. Here, we report on the ultra-large-scale synthesis of monodisperse nanocrystals using inexpensive and non-toxic metal salts as reactants. We were able to synthesize as much as 40 g of monodisperse nanocrystals in a single reaction, without a size-sorting process. Moreover, the particle size could be controlled simply by varying the experimental conditions. The current synthetic procedure is very general and nanocrystals of many transition metal oxides were successfully synthesized using a very similar procedure.
New technologies for imaging molecules, particularly optical technologies, are increasingly being used to understand the complexity, diversity and in vivo behaviour of cancers. 'Omic' approaches are providing comprehensive 'snapshots' of biological indicators, or biomarkers, of cancer, but imaging can take this information a step further, showing the activity of these markers in vivo and how their location changes over time. Advances in experimental and clinical imaging are likely to improve how cancer is understood at a systems level and, ultimately, should enable doctors not only to locate tumours but also to assess the activity of the biological processes within these tumours and to provide 'on the spot' treatment.
We report an innovative approach to producing bacteriochlorins (bacs) via formal cycloaddition by subjecting a porphyrin to a trimolecular reaction. Bacs are near-infrared probes with the intrinsic ability to serve in multimodal imaging. However, despite their ability to fluoresce and chelate metal ions, existing bacs have thus offered limited ability to label biomolecules for target specificity or have lacked chemical purity, limiting their use in bio-imaging. In this work, bacs allowed a precise and controlled appending of clickable linkers, lending the porphyrinoids substantially more chemical stability, clickability, and solubility, rendering them more suitable for preclinical investigation. Our bac probes enable the targeted use of biomolecules in fluorescence imaging and Cerenkov luminescence for guided intraoperative imaging. Bacs' capacity for chelation provides opportunities for use in non-invasive positron emission tomography/computed tomography. Herein, we report the labeling of bacs with Hs1a, a (NaV1.7)-sodium-channel-binding peptide derived from the Chinese tarantula Cyriopagopus schmidti to yield Bac-Hs1a and radiolabeled Hs1a, which shuttles our bac sensor(s) to mouse nerves. In vivo, the bac sensor allowed us to observe high signal-to-background ratios in the nerves of animals injected with fluorescent Bac-Hs1a and radiolabeled Hs1a in all imaging modes. This study demonstrates that Bac-Hs1a and [64Cu]Cu-Bac-Hs1a accumulate in peripheral nerves, providing contrast and utility in the preclinical space. For the chemistry and bio-imaging fields, this study represents an exciting starting point for the modular manipulation of bacs, their development and use as probes for diagnosis, and their deployment as formidable multiplex nerve-imaging agents for use in routine imaging experiments.
Recently, supramolecular chemistry with its unique properties has received considerable attention in many fields. Supramolecular fluorescent systems constructed on the basis of macrocyclic hosts are not only effective in overcoming the limitations of imaging and diagnostic reagents, but also in enhancing their performances. This paper summarizes the recent advances in supramolecular fluorescent systems based on host-guest interactions and their application in bioimaging and therapy as well as the challenges and prospects in developing novel supramolecular fluorescent systems.
Various functional nanomaterials have been fabricated as diagnostic and therapeutic nanomedicines; however, the nanoparticles closely interact with proteins when immersed in biological fluids, forming a "protein corona" that critically alters the biological identity of nanomedicine. Here, we developed a robust strategy to construct theranostic nanoprobes based on protein-corona-coated Fe3O4 nanoparticles and biomineralization in the corona. Water-soluble carboxylic Fe3O4 nanoparticles were prepared by treating oleate-capped Fe3O4 nanoparticles with Lemieux-von Rudloff reagent. Bovine serum albumin (BSA) was used as a model protein to form a corona on the surface of Fe3O4 nanoparticles, endowing the Fe3O4 nanoparticles with biocompatibility and nonimmunogenicity. The protein corona also provides a template for biomimetic mineralization of Fe3+ with tannic acid (TA) to construct Fe3O4@BSA-TAFeIII nanoprobes. The TA-Fe(III) biominerals can not only act as photothermal therapy agents but also interact with unsaturated transferrin in plasma to form a "hybrid" corona, enabling the nanoprobes to target tumor cells through the mediation of transferrin receptors, which commonly overexpress on tumor cell membranes. Once taken in by tumor cells, the protonation of phenol hydroxyl groups in acidic lysosomes would lead to the release of Fe3+, inducing tumor cell death through a ferroptosis/apoptosis hybrid pathway. In addition, the released Fe3+ can boost the T1-weighted MR imaging performance, and the Fe3O4 nanoparticles serve as T2-weighted MR imaging contrast agents. It is thus believed that the current nanoprobes can realize the enhanced dual-modality MR imaging and combined therapy of tumors through controlling the protein corona and biomineralization.
In contrast to regular J- and H-aggregates, thin film squaraine aggregates usually have broad absorption spectra containing both J-and H-like features, which are favorable for organic photovoltaics. Despite being successfully applied in organic photovoltaics for years, a clear interpretation of these optical properties by relating them to specific excited states and an underlying aggregate structure has not been made. In this work, by static and transient absorption spectroscopy on aggregated n-butyl anilino squaraines, we provide evidence that both the red- and blue-shifted peaks can be explained by assuming an ensemble of aggregates with intermolecular dipole-dipole resonance interactions and structural disorder deriving from the four different nearest neighbor alignments─in sharp contrast to previous association of the peaks with intermolecular charge-transfer interactions. In our model, the next-nearest neighbor dipole-dipole interactions may be negative or positive, which leads to the occurrence of J- and H-like features in the absorption spectrum. Upon femtosecond pulse excitation of the aggregated sample, a transient absorption spectrum deviating from the absorbance spectrum emerges. The deviation finds its origin in the excitation of two-exciton states by the probe pulse. The lifetime of the exciton is confirmed by the band integral dynamics, featuring a single-exponential decay with a lifetime of 205 ps. Our results disclose the aggregated structure and the origin of red- and blue-shifted peaks and explain the absence of photoluminescence in squaraine thin films. Our findings underline the important role of structural disorder of molecular aggregates for photovoltaic applications.
Multimodality imaging recognized as a promising monitoring strategy can serve the needs of accurate diagnosis and treatment of cancer by providing molecular and anatomic information about tumor sites. However, the probes based on multiple imaging modalities for surgery navigation remain limited due to poor biocompatibility and tumor targeting specificity. Herein, we present a small-molecule near-infrared fluorescence/magnetic resonance (NIRF/MR) imaging probe, Gd-NMC-3, covalently coupled with DCDSTCY and Gd-DOTA via butane diamine, for precise detection and intraoperative visualization. The in vitro and in vivo studies demonstrated that Gd-NMC-3 could be effectively accumulated in tumor sites as a bimodal imaging molecule exhibiting significant fluorescence accumulation and reasonable relaxation property in tumors with low cytotoxicity and good biocompatibility. Furthermore, Gd-NMC-3 was successfully applied to provide real-time visual navigation in LM3 orthotopic and subcutaneous tumor models to guide the resection of tumors. Importantly, no more fluorescence was observed in mice after operation, implying the total removal of tumor tissues. In conclusion, Gd-NMC-3 has great potential to be applied in the clinic based on its high resolution and sensitivity in tumor imaging.
The rhodamine appended Fe(III)-catecholate complex Fe(RhoCat) 3 is reported as a smart dual-modal T 1 MRI-optical imaging probe. The high spin Fe(III) coordination sphere and rhodamine unit act as MRI and optical reporters respectively. The probe showed r 1-relaxivity of 4.37 mM-1 s-1 at 1.41 T via the interaction of second sphere water molecules to coordinated oxygen atoms. It produced an enhanced signal intensity of phantom images at 7.0 T animal research MRI/MRS scanner at 25 C and pH 7.3. The interaction of the probe with bovine serum albumin (BSA) significantly improved r 1-relaxivity (7.09 mM-1 s-1). Moreover, the optical imaging reporter rhodamine moiety has exhibited sensitivity towards biomolecule nitric oxide (NO) and acidic pH via the formation of ring-opened tautomer of rhodamine, wherein the r 1-relaxivity of the probe enhanced to 5.19 mM-1 s-1 for NO and slightly decreased for acidic pH. Further, the probe has visualized NO in adenocarcinoma gastric (AGS) cells via a turn-on fluorescence mechanism with 80% cell viability. Thus, Fe(RhoCat) 3 is demonstrated as a potential dual "MRI-ON and Fluorescence-ON" molecular imaging probe to visualize the NO molecule and acidic pH in the tumour microenvironment.
Stimuli-responsive in situ self-assembly of small molecules to form nanostructures in living subjects has produced promising tools for molecular imaging and tissue engineering. However, controlling the self-assembly process to simultaneously activate multimodality imaging signals in a small-molecule probe is challenging. In this paper, we rationally integrate a fluorogenic reaction into enzyme-responsive in situ self-assembly to design small-molecule-based activatable near-infrared (NIR) fluorescence and magnetic resonance (MR) bimodal probes for molecular imaging. Using alkaline phosphatase (ALP) as a model target, we demonstrate that probe (P-CyFF-Gd) can be activated by endogenous ALP overexpressed on cell membranes, producing membrane-localized assembled nanoparticles (NPs) that can be directly visualized by cryo-SEM. Simultaneous enhancements in NIR fluorescence (>70-fold at 710 nm) and r1 relaxivity (∼2.3-fold) enable real-time, high-sensitivity, high-spatial-resolution imaging and localization of the ALP activity in live tumor cells and mice. P-CyFF-Gd can also delineate orthotopic liver tumor foci, facilitating efficient real-time, image-guided surgical resection of tumor tissues in intraoperative mice. This strategy combines activatable NIR fluorescence via a fluorogenic reaction and activatable MRI via in situ self-assembly to promote ALP activity imaging, which could be applicable to design other activatable bimodal probes for in vivo imaging of enzyme activity and locations in real time.
Tens of millions of contrast-enhanced magnetic resonance imaging (MRI) exams are performed annually around the world. The contrast agents, which improve diagnostic accuracy, are almost exclusively small, hydrophilic gadolinium(III) based chelates. In recent years concerns have arisen surrounding the long-term safety of these compounds, and this has spurred research into alternatives. There has also been a push to develop new molecularly targeted contrast agents or agents that can sense pathological changes in the local environment. This comprehensive review describes the state of the art of clinically approved contrast agents, their mechanism of action, and factors influencing their safety. From there we describe different mechanisms of generating MR image contrast such as relaxation, chemical exchange saturation transfer, and direct detection and the types of molecules that are effective for these purposes. Next we describe efforts to make safer contrast agents either by increasing relaxivity, increasing resistance to metal ion release, or by moving to gadolinium(III)-free alternatives. Finally we survey approaches to make contrast agents more specific for pathology either by direct biochemical targeting or by the design of responsive or activatable contrast agents.
This article describes the fabrication of nanosized magneto-vesicles (MVs) comprising tunable layers of densely-packed superparamagnetic iron oxide nanoparticles (SPIONs) in membranes via cooperative assembly of polymer-tethered SPIONs and free poly(styrene)-b-poly(acrylic acid) (PS-b-PAA). The membrane thickness of MVs could be well controlled from 9.8 to 93.2 nm by varying the weight ratio of PS-b-PAA to SPIONs. The increase in membrane thickness was accompanied with the transition from monolayer MVs, to double-layered MVs and to multilayered MVs (MuMVs). This can be attributed to the variation in the hydrophobic/hydrophilic balance of polymer-grafted SPIONs upon the insertion and binding of PS-b-PAA onto the surface of nanoparticles. Therapeutic agents can be efficiently encapsulated in the hollow cavity of MVs and the release of payload can be tuned by varying the membrane thickness of nanovesicles. Due to the high packing density of SPIONs, the MuMVs showed the highest magnetization and transverse relaxivity rate (r2) in magnetic resonance imaging (MRI) among these MVs and individual SPIONs. Upon intravenous injection, doxorubicin-loaded MuMVs conjugated with RGD peptides could be effectively enriched at tumor sites due to synergetic effect of magnetic and active targeting. As a result, they exhibited drastically enhanced signal in MRI, improved tumor delivery efficiency of drugs as well as enhanced antitumor efficacy, compared with groups with only magnetic or active targeting strategy. The unique nanoplatform may find applications in effective disease control by delivering imaging and therapy to organs/tissues that are not readily accessible by conventional delivery vehicles.
Chemistry welcomes a new bond: The mechanical bond has endowed molecules with component parts whose movements can be controlled and monitored. In his Nobel Lecture, J. F. Stoddart describes how being able to template the formation of mechanically interlocked molecules has led to the design and synthesis of shuttles, switches, and machines at the nanoscale.
Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.
Squaraine rotaxanes are mechanically interlocked molecules comprised of a dumbbell shaped squaraine dye inside a tetralactam macrocycle. Previous squaraine rotaxanes have employed planar squaraine dyes with 4-aminophenyl, 2-aminothiophene or N-amino units appended to the central C4O2 core. Here we describe two rotaxanes that encapsulate a 3,3-dimethylindoline squaraine inside a tetralactam with anthracene sidewalls. The rotaxanes were prepared by a templated clipping reaction and an X-ray crystal structure shows that the squaraine gem-dimethyl groups force a relatively wide separation between the macrocycle anthracene sidewalls. The decreased interaction between the encapsulated squaraine and the anthracene sidewalls leads to a smaller red-shift of the squaraine absorption and emission bands. Solution-state studies show that the gem-dimethyl groups in 3,3-dimethylindoline squaraine dyes are large enough to prevent macrocyle threading or rotaxane unthreading. One of the new rotaxanes emits orange light (560-650 nm) and there is a ten-fold enhancement in squaraine fluorescence quantum yield upon encapsulation as a rotaxane. This orange-emitting dye completes the palette of known squaraine rotaxane fluorophores whose emission profiles span the color range from green to near-infrared.
Simultaneous positron emission tomography and MRI (PET/MRI) is a technology that combines the anatomic and quantitative strengths of MR imaging with physiologic information obtained from PET. PET and computed tomography (PET/CT) performed in a single scanning session is an established technology already in widespread and accepted use worldwide. Given the higher cost and complexity of operating and interpreting the studies obtained on a PET/MRI system, there has been question as to which patients would benefit most from imaging with PET/MRI versus PET/CT. In this article, we compare PET/MRI with PET/CT, detail the applications for which PET/MRI has shown promise and discuss impediments to future adoption. It is our hope that future work will prove the benefit of PET/MRI to specific groups of patients, initially those in which PET/CT and MRI are already performed, leveraging simultaneity and allowing for greater degrees of multiparametric evaluation.
Level of evidence:
5 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2017;46:1247-1262.
In vivo imaging, which enables us to peer deeply within living subjects, is producing tremendous opportunities both for clinical diagnostics and as a research tool. Contrast material is often required to clearly visualize the functional architecture of physiological structures. Recent advances in nanomaterials are becoming pivotal to generate the high-resolution, high-contrast images needed for accurate, precision diagnostics. Nanomaterials are playing major roles in imaging by delivering large imaging payloads, yielding improved sensitivity, multiplexing capacity, and modularity of design. Indeed, for several imaging modalities, nanomaterials are now not simply ancillary contrast entities, but are instead the original and sole source of image signal that make possible the modality's existence. We address the physicochemical makeup/design of nanomaterials through the lens of the physical properties that produce contrast signal for the cognate imaging modality-we stratify nanomaterials on the basis of their (i) magnetic, (ii) optical, (iii) acoustic, and/or (iv) nuclear properties. We evaluate them for their ability to provide relevant information under preclinical and clinical circumstances, their in vivo safety profiles (which are being incorporated into their chemical design), their modularity in being fused to create multimodal nanomaterials (spanning multiple different physical imaging modalities and therapeutic/theranostic capabilities), their key properties, and critically their likelihood to be clinically translated.
Multiple imaging modalities are often required for in vivo imaging applications that require both high probe sensitivity and excellent spatial and temporal resolution. In particular, MR and optical imaging are an attractive combination that can be used to determine both molecular and anatomical information. Herein, we describe the synthesis and in vivo testing of two multimeric NIR-MR contrast agents that contain three Gd(III) chelates and an IR-783 dye moiety. One agent contains a PEG linker and the other a short alkyl linker. These agents label cells with extraordinary efficacy and can be detected in vivo using both imaging modalities. Biodistribution of the PEGylated agent shows observable fluorescence in xenograft MCF7 tumors and renal clearance by MR imaging.
A protease-activated ratiometric fluorescent probe based on fluorescence resonance energy transfer between a pH-sensitive fluorescent dye and biocompatible Fe3O4 nanocrystals was constructed. A peptide substrate of MMP-9 served as a linker between the particle quencher and the chromophore that was covalently attached to the antitumor antibody. The optical response of the probe to activated MMP-9 and gastric cell line SGC7901 tumor cells was investigated, followed by in vivo tumor imaging. Based on the ratiometric pH response to the tumor microenvironment, the resulting probe was successfully used to image the pH of subcutaneous tumor xenografts.
A water-soluble T1 magnetic resonance imaging contrast agent (1) has been synthesized. The bimodal contrast agent 1 responds to the Cu(2+) ion in living cells by enhancing the MRI modality signal whereas the optical signal gradually drops. This dual modality probe response depends on the cellular free copper ions in RAW 264.7 cells even at the micromolar level.
A new squaraine rotaxane molecular shuttle exhibits high chemical stability and acts as a deep-red, fluorescent and colorimetric sensor for Cl− anion with reversible, ratiometric response. The molecular design encapsulates a dihydroxyl substituted squaraine dye inside an anthracene-containing tetralactam macrocycle and a “clicked capping” reaction was used to convert an appropriate pseudorotaxane precursor into a permanently interlocked rotaxane in high yield. Reversible binding of Cl− to the rotaxane in solution, or on the surface of prototype dipsticks, causes lateral displacement of the surrounding macrocycle away from the central squaraine station and a substantial 30–40 nm shift in the squaraine absorption/fluorescence maxima that can be easily detected by the naked eye. The collective attributes of intense absorption/emission and ratiometric response at deep-red wavelengths is a significant advance in optical Cl− sensor performance by an organic molecule.
We have designed, prepared, and tested a new set of multidentate catechol- and polyethylene glycol (PEG)-derivatized oligomers, OligoPEG-Dopa, as ligands that exhibit strong affinity to iron oxide nanocrystals. The ligands consist of a short poly(acrylic acid) backbone laterally appended with several catechol anchoring groups and several terminally functionalized PEG moieties to promote affinity to aqueous media and to allow further coupling to target molecules (bio and others). These multicoordinating PEGylated oligomers were prepared using a relatively simple chemical strategy based on N,N'-dicyclohexylcarbodiimide (DCC) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) condensation. The ability of these catechol-functionalized oligomers to impart long-term colloidal stability to the nanoparticles is compared to other control ligands, namely, oligomers presenting several carboxyl groups and monodentate ligands presenting either one catechol or one carboxyl group. We found that the OligoPEG-Dopa ligands provide rapid ligand exchange, and the resulting nanoparticles exhibit greatly enhanced colloidal stability over a broad pH range and in the presence of excess electrolytes; stability is notably improved compared to non-catechol presenting molecular or oligomer ligands. By inserting controllable fractions of azide-terminated PEG moieties, the nanoparticles (NPs) become reactive to complementary functionalities via azide-alkyne cycloaddition (Click), which opens up the possibility of biological targeting of such stable NPs. In particular, we tested the Click coupling of azide-functionalized nanoparticles to an alkyne-modified dye. We also measured the MRI T(2) contrast of the OligoPEG-capped Fe(3)O(4) nanoparticles and applied MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to test the potential cytotoxicity of these NPs to live cells; we found no measurable toxicity to live cells.
Advances in our understanding of the genetic basis of disease susceptibility coupled with prominent successes for molecular targeted therapies have resulted in an emerging strategy of personalized medicine. This approach envisions risk stratification and therapeutic selection based on an individual’s genetic makeup and physiologic state (the latter assessed through cellular or molecular phenotypes). Molecularly targeted nanoparticles can play a key role in this vision through noninvasive assessments of molecular processes and specific cell populations in vivo, sensitive molecular diagnostics, and targeted delivery of therapeutics.
The current lack of suitable probes has limited the in vivo imaging of reactive oxygen/nitrogen species (ROS/RNS). ROS/RNS are often generated by ischemia-induced inflammation; defining the extent of tissue involvement or ROS/RNS-related damage would have a significant clinical impact. We present the preparation and demonstration of a fluorogenic sensor for monitoring peroxynitrite (ONOO(-)) and myeloperoxidase (MPO) mediated hypochlorous acid (HOCl/OCl(-)) production. The sensor consists of a long circulating biocompatible nanoparticle that targets phagocytic cells in vivo and is coated with approximately 400 quenched oxazine fluorophores that are released by reaction with HOCl or ONOO(-) but are stable toward oxidants such as hydroxyl radical, hydrogen peroxide, and superoxide. MPO-dependent probe activation is chloride ion dependent and is negated in flow cytometry studies of MPO inhibitor treated neutrophils. Fluorescence reflectance imaging and microscopic fluorescence imaging in mouse hearts after myocardial infarction showed probe release into neutrophil-rich ischemic areas, making this ROS/RNS sensor a novel prognostic indicator.
The design of a sensor array that uses a single entity as both the host and the indicator (squaraine dye, SQ) to differentiate a series of metal ions and a series of thiols is reported. The metal ions and thiols act as both analytes and "modulators" of the squaraine response allowing pattern-based discrimination. Mercury(II), palladium(II), copper(II), iron(II), and nickel(II) can be discriminated when combining SQ with five thiols: propane thiol (PT), 3-mercaptopropionic acid (MPA), naphthalene-2-thiol (NT), 2,3-dimercaptopropanol (DMP), and 2-acetylamino-3-mercaptopropionic acid methyl ester (ACM). Likewise, the five thiols can be discriminated using SQ and the five metals. For example, SQ in combination with 2-acetylamino-3-mercaptopropionic acid methyl ester (ACM) afforded very good differentiation of all five metal ions. However, propanethiol, 3-mercaptopropionic acid, and naphthalene-2-thiol produced very similar differentiation of the considered metal ions. On the other hand, all metal ions considered in this study are able to discriminate 2,3-dimercaptopropanol (DMP) and 2-acetylamino-3-mercaptopropionic acid methyl ester (ACM) clearly and completely, both from one another and from the other three thiols (PT, NT, MPA). Importantly, mercury(II) is the only metal ion able to effect the discrimination of naphthalenethiol (NT) from PT and MPA, thus giving the best discrimination overall. The study shows that complex discrimination of widely diverse classes, metal ions and thiols, can be achieved via a single receptor/indicator.
The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.
The design of near-infrared fluorescent (NIRF) probes that are activated by specific proteases has, for the first time, allowed enzyme activity to be imaged in vivo. In the current study, we report on a method of imaging enzyme activity using two fluorescent probes that, together, provide improved quantitation of enzymatic activity. The method employs two chemically similar probes that differ in their degradability by cathepsin B. One probe consists of the NIRF dye Cy5.5 attached to a particulate carrier, a crosslinked iron oxide nanoparticle (CLIO), through cathepsin B cleavable L-arginyl peptides. A second probe consists of Cy3.5 attached to a CLIO through proteolytically resistant D-arginyl peptides. Using mixtures of the two probes, we have shown that the ratio of Cy5.5 to Cy3.5 fluorescence can be used to determine levels of cathepsin B in the environment of nanoparticles with macrophages in suspension. After intravenous injection, tissue fluorescence from the nondegradable Cy3.5-D-arginyl probe reflected nanoparticle accumulation, while fluorescence of the Cy5.5-L-arginyl probe was dependent on both accumulation and activation by cathepsin B. Dual wavelength ratio imaging can be used for the quantitative imaging of a variety of enzymes in clinically important settings, while the magnetic properties of the probes allow their detection by MR imaging.
Anthracene-containing tetralactam macrocycles are prepared and found to have an extremely high affinity for squaraine dyes in chloroform (log Ka = 5.2). Simply mixing the two components produces highly fluorescent, near-infrared inclusion complexes in quantitative yield. An X-ray crystal structure shows the expected hydrogen bonding between the squaraine oxygens and the macrocycle amide NH residues, and a high degree of cofacial aromatic stacking. The kinetics and thermodynamics of the assembly process are very sensitive to small structural changes in the binding partners. For example, a macrocycle containing two isophthalamide units associates with the squaraine dye in chloroform 400,000 times faster than an analogous macrocycle containing two 2,6-dicarboxamidopyridine units. Squaraine encapsulation also occurs in highly competitive media such as mixed aqueous/organic solutions, vesicle membranes, and the organelles within living cells. The highly fluorescent inclusion complexes possess emergent properties; that is, as compared to the building blocks, the complexes have improved chemical stabilities, red-shifted absorption/emission maxima, and different cell localization propensities. These are useful properties for new classes of near-infrared fluorescent imaging probes.
Molecular Magnetic Resonance Imaging with Gd(III)-Based Contrast Agents: Challenges and Key Advances
- H Li
- T J Meade
Li, H.; Meade, T. J. Molecular Magnetic Resonance Imaging
with Gd(III)-Based Contrast Agents: Challenges and Key Advances. J.
Am. Chem. Soc. 2019, 141, 17025−17041.