PFCE is a highly sensitive 19F MRI contrast agent due to its 20 magnetically equivalent 19F atoms, enabling effective detection of fluorine-loaded cells in inflammatory processes (49). However, its extremely long biological half-life (>250 days) limits clinical applicability. Compared with other PFCs such as PFOB, PFCE can deliver stronger signal intensities but often poses prolonged organ retention. Investigations into alternative PFCs with shorter half-lives identified PFD, PFOB, and F-44E, with murine liver and spleen half-lives of 9, 12, and 28 days, respectively (61,62). Among these, PFOB emerged as a promising candidate for clinical translation, providing 37% of PFCE's signal intensity in inflammation imaging models (61,63,64).

To overcome the limitations of PFCE and enhance its targeting capabilities, researchers synthesized c-Met-targeting peptide-functionalized PFCE nanoparticles (AH111972-PFCE NPs) with a particle size of 89.3±17.8 nm. These NPs exhibited high specificity and strong c-Met-targeting ability, enabling precise detection of small colorectal liver metastases, particularly ill-defined fused metastases undetectable by ¹H-MRI, with ultralong tumor retention of at least 7 days and minimal side effects (65). Similarly, PFCE encapsulated in PLGA-PEG-mannose nanoparticles targeted tumor-associated macrophages overexpressing the mannose receptor (MRC1/CD206), facilitating in vivo imaging of the tumor microenvironment by 19F MRI. At 48 h post-injection, nanoparticle retention at the tumor site was confirmed, benefiting from robust and specific 19F signals due to the lack of background 19F in the body (66).

Moreover, PFCE demonstrates superior biocompatibility compared with other fluorine reporter probes such as HFB for tissue oxygenation assessment (67,68). Unlike HFB, which induces tissue necrosis and mobility limitations compromising extended pO₂ measurements, PFCE exhibits no muscle tissue toxicity and does not affect animal behavior ≤36 h post-injection, allowing accurate and prolonged assessment (67,68). Collectively, these studies highlight the potential of PFCE in 19F MRI applications when strategies are employed to enhance targeting and biocompatibility while mitigating its prolonged biological half-life, thereby advancing its clinical suitability (29,37,63,64).

To further improve the utility of PFCE as a 19F MRI contrast agent, a highly concentrated and stable colloidal nanoemulsion (NE) was developed using the semifluorinated triblock copolymer M2F8H18 to encapsulate PFCE at 35% v/v, enhancing imaging sensitivity for in vivo cancer detection (69,70). The resulting NE nanoparticles mean value 210±38 nm in size with a polydispersity index of 0.03, exhibiting long-term stability of at least 98 days at 4°C and maintaining stability at physiological temperatures and in serum, thus preventing particle growth that could lead to embolism. In vitro cytotoxicity assays using 4T1-Luc murine breast carcinoma cells showed negligible cell death, even at high PFCE concentrations ≤20 mg/ml and incubation periods ≤48 h, indicating good biocompatibility. In a tumor-bearing mouse model, intravenous administration of the NE resulted in high 19F MRI signals with signal-to-noise ratios ≤100 in clinically relevant scan times (~11 min) (71). The NE circulated stably in the vasculature, with visible accumulation in the heart and inferior vena cava at 6 h post-injection, and accumulated in tumors with an estimated concentration of 4-9×10¹⁷ 19F spins per voxel. The PFCE signal persisted at the tumor site ≤14 days post-injection, with 50% remaining at Day 7 and 33% at Day 14, demonstrating prolonged tumor imaging capability. The high PFCE loading and passive targeting through the enhanced permeability and retention (EPR) effect enabled enhanced 19F MRI contrast and precise, prolonged tumor imaging. However, the nanoparticle size led to uptake by the mononuclear phagocyte system (MPS), resulting in accumulation in the liver and spleen, which may limit specificity. Further optimization, such as reducing particle size, is necessary to minimize MPS uptake. This novel, highly stable NE formulation with unprecedented PFCE loading and prolonged in vivo stability offers significant potential as a 19F MRI contrast agent for cancer diagnostics.

Due to its favorable NMR properties and biocompatibility, PFOB has been extensively studied as a 19F MRI contrast agent. Compared with PFCE, PFOB offers a shorter biological half-life and thus lower long-term organ retention, although this advantage comes at the cost of lower signal intensity. Recent efforts to enhance the imaging efficacy and targeting capabilities of PFOB led to the synthesis of PLGA-PEG nanocapsules encapsulating a liquid PFOB core via an emulsion-evaporation process (72,73). Incorporating PEG through PLGA-PEG diblock copolymers enhanced PFOB encapsulation efficiency compared with plain PLGA nanocapsules, as estimated by 19F NMR spectroscopy (74). The PEGylated nanocapsules (mean value 120 nm in diameter) maintained a spherical core-shell morphology confirmed by dynamic light scattering, transmission electron microscopy and scanning electron microscopy analyses. PEGylation was confirmed by zeta potential measurements and X-ray photoelectron spectroscopy, resulting in reduced complement activation in vitro, indicative of stealth properties. In vivo 19F MRI studies in mice demonstrated that PEGylated nanocapsules accumulated in CT26 xenograft tumors 7 h post-intravenous injection, while plain nanocapsules were undetectable. This highlights the efficacy of PEGylation in prolonging circulation time and enhancing tumor targeting via the EPR effect. However, liver accumulation was still observed, indicating a need for further optimization to reduce off-target uptake (72).

Optimizing MRI pulse sequences has also been explored to improve the sensitivity of PFOB-based imaging. Selecting appropriate bandwidths of 180° pulses in spin-echo sequences mitigated detrimental effects of J-coupling, enhancing detection of the CF3 resonance (75). The T2 relaxation time of the CF3 group depended on the interpulse delay in multispin-echo sequences; optimizing this delay yielded an imaging sequence with superior sensitivity over traditional gradient echo and chemical shift imaging sequences. However, the efficacy of this approach relies on precise control of interpulse delays and pulse bandwidths, posing practical challenges in clinical settings (75).

In cell therapies, non-invasive tracking of transplanted cells via 19F MRI is crucial due to its high specificity and negligible background signal (76). A novel nano-contrast agent, termed PSS-NP, was formulated with a PFOB core encapsulated within a PLGA shell and coated with polystyrene sulfonate (PSS) to enhance uptake by MSCs through caveolae-mediated endocytosis. PSS-NPs exhibited a hydrodynamic size of ~140 nm with a zeta potential of −60 mV, indicating good stability, and were efficiently internalized by MSCs without affecting proliferation or osteoblastic differentiation potential, as confirmed by flow cytometry, confocal microscopy, and alkaline phosphatase activity assays (77). In vitro, PSS-NP-labelled cells maintained a detectable 19F MRI signal for ≤14 days. In vivo, these cells could be tracked using 19F MRI for ≤two months post-transplantation in mice, retaining their ability to form mineralized tissue. Importantly, PSS-NP-labelled cells enabled monitoring of immune rejection, evidenced by a 40% loss of 19F MRI signal one week after transplantation in immunocompetent BALB/c mice compared with a 10% loss in immunocompromised NOD/SCID mice. This work demonstrated a safe and efficient method for stem cell labelling that provides insights into cell survival and immune rejection in vivo. A remaining challenge is the reliance on a high-field 16.4 T MRI scanner, which is not directly applicable to clinical settings typically employing 1.5 T to 3 T MRI, necessitating further research for clinical translation (76).

Novel MRI contrast agents play a crucial role in enhancing the precision of cancer therapies, particularly in MRI-guided radiotherapy (RT). Researchers developed pH and O₂ dual-sensitive nano-molecular imaging probes (Gly-PFOBs) based on PFOB and glycerol-weighted CEST, exhibiting dual 19F/1H-CEST MRI (Fig. 2) (78). The hypothesis was that Gly-PFOBs could ameliorate tumor hypoxia by delivering oxygen and serve as MRI contrast agents to optimize the radiotherapy time window. The Gly-PFOBs demonstrated excellent pH and oxygen sensitivity in vitro, with CEST signal intensity changes corresponding to pH variations from 5.9-8.0 and oxygen concentration adjustments. In vivo studies using NCI-H460 lung cancer xenograft models showed that oxygenated Gly-PFOBs effectively improved tumor oxygenation, as evidenced by blood oxygen level-dependent MRI and enhanced RT efficacy (78). Specifically, the tumor growth inhibition rate in mice treated with RT and Gly-PFOBs at the optimized time window (1-2 h post-injection) was 81.31%, markedly higher than RT alone (44.72%). The probes exhibited superior therapeutic efficacy and biosafety, with no significant toxicity observed in major organs.

PFPEs have emerged as promising 19F MRI contrast agents for noninvasive in vivo cell tracking due to their biocompatibility. Compared with PFCE or PFOB, PFPE agents can be formulated in a variety of structures that influence mobility and relaxation properties, offering flexibility in optimizing imaging performance. 19F MRI enables selective imaging of PFPE-labeled cells against a background-free environment, since biological tissues lack mobile fluorine atoms, while 1H-MRI provides anatomical context (70). In a pioneering study (79), dendritic cells (DCs) labeled ex vivo with PFPEs retained their function, allowing in vivo tracking of DC migration in mice via 19F MRI (49,79). Antigen-specific T cells labeled with PFPE nanoemulsions were similarly monitored over 21 days, revealing dynamic patterns in lymph nodes and enabling quantification of apparent T-cell numbers, though in vivo cell division complicated accuracy (80,81). To enhance detection, dual-mode PFPE nanoemulsions conjugated with fluorescent dyes (FITC, Alexa647, BODIPy-TR) were developed, facilitating simultaneous 19F MRI and fluorescence detection (67). These nanoemulsions (<200 nm) were rapidly internalized by various cells, and the proportional relationship between intracellular fluorescence and 19F NMR signal enabled calibration of cell loading, improving analysis via fluorescence microscopy and fluorescence-activated cell sorting.

Designing effective polymeric 19F MRI contrast agents is challenging due to the hydrophobicity of fluorine, leading to aggregation and signal attenuation. To address this, thermoresponsive PFPE-based comb-shaped poly(2-oxazoline) s with varying side-chain structures were investigated (82). At increased temperatures, some polymers transitioned to unimers, enhancing imaging intensity, while others formed aggregates, degrading performance, underscoring the importance of polymer self-association in designing smart, thermoresponsive 19F MRI agents (83).

The effect of different hydrophilic segments on imaging performance was further studied using PFPE-containing amphiphilic block copolymers synthesized via RAFT polymerization (84). Block copolymers PMSEA-PFPE, POEGA-PFPE, and POMOXA-PFPE, prepared from hydrophilic monomers MSEA, OEGA, and OMOXA, respectively, formed assemblies with hydrophobic PFPE cores and hydrophilic shells, as confirmed by dynamic light scattering and molecular dynamics simulations. Simulations revealed that POMOXA's rigid, extended chains led to shorter 19F NMR spin-spin relaxation times (T₂=33.9 msec), lower 19F spin visibility (59%), and weaker MRI signals [signal-to-noise ratio (SNR)=36.9 at 6.8 mg/ml fluorine concentration]. By contrast, PMSEA-PFPE exhibited higher 19F spin visibility (100%) and stronger MRI signal intensity (SNR=222.0 at 6.8 mg/ml), despite a shorter T2 (41.3 msec) than POEGA-PFPE (62.3 msec). These results emphasize that both high fluorine visibility and long T2 relaxation times are crucial for effective contrast agents, highlighting the significance of hydrophilic segments in influencing self-assembly, chain mobility, and NMR properties.

These studies collectively demonstrate the versatility and potential of PFPE-based 19F MRI agents in noninvasive cell tracking and advancing cellular therapeutics (50,67,85-87). Limitations include quantification challenges due to in vivo cell division and the need to optimize polymer designs to prevent aggregation that diminishes imaging quality. Further in vivo evaluations are necessary to assess imaging efficacy and biocompatibility, as some studies remain at the in vitro or simulation level.

Polymer-based 19F MRI contrast agents are promising tools for non-invasive, targeted imaging due to the unique magnetic properties and negligible background signals of fluorine in biological systems. Compared with PFC-based agents, which often exhibit very high 19F loading but potential issues with half-life or RES uptake, polymeric agents can be tailored to balance signal intensity, biocompatibility, and circulation times (33). To enhance imaging performance, various branched and hyperbranched fluorinated polymers have been developed. Fu et al (88) synthesized branched fluorinated glycopolymers via one-pot RAFT polymerization, incorporating glucose units and disulfide cross-linkers. These polymers effectively targeted cancer cells through interactions with overexpressed sugar transporters and showed enhanced 19F MRI signals in reductive environments typical of cancer cells. Similarly, segmented highly branched polymers (SHBPs) composed of fluorinated and PEG-based monomers were reported using RAFT-mediated self-condensing vinyl copolymerization (89). SHBPs with statistical copolymeric acrylate segments exhibited a single 19F signal, long T2 relaxation times, and high fluorine content, making them excellent imaging candidates. A hyperbranched poly(N,N-dimethylacrylamide) conjugated with 5-fluorouracil via an enzyme-degradable peptide linker demonstrated enzyme-responsive 5-FU release, resulting in significant changes in T₂19F NMR/MRI relaxation times and enabling monitoring of drug release (90).

Modification of natural polysaccharides such as alginic acid, dextran, and polygalacturonic acid with 3-aminobenzotrifluoride produced water-soluble materials containing 1-14% fluorine (91). These materials exhibited low toxicity, lysosomal localization and rapid renal clearance, suggesting suitability for imaging gastrointestinal or genitourinary systems. An injectable 19F-labeled hyaluronic acid hydrogel formed via carbazone reaction was monitored by both ¹H and 19F MRI without affecting mechanical properties, indicating potential for minimally invasive, biocompatible tracking (92,93). Superfluorinated polyphosphazene (PPz) polymers modified with sodium mercaptoethanesulfonate achieved exceptional water solubility (>360 mg/ml) and substantial MRI signal enhancement compared with aqueous trifluoroacetic acid controls, showing promise as future MRI contrast agents (94). However, limitations in some perfluorocarbon-based materials necessitate continued optimization for clinical applications (94).

To address these challenges, a novel hyperbranched polymeric 19F MRI contrast agent based on β-cyclodextrin and phosphorylcholine was developed to enhance T2 relaxation time and fluorine content (95,96). Higher branching degrees and optimal fluorine loading prolonged T2 relaxation times, achieving a maximum fluorine content of 11.85% and a T2 relaxation time of ≤612 msec at 9.4 T. In vitro cytotoxicity assays showed excellent biocompatibility against HUVEC and 4T1 cells, with viability exceeding 80% at concentrations ≤1,000 μg/ml (37). In vivo studies indicated no significant tissue damage in mice (39,95,96). This contrast agent provided high-performance 19F MRI capabilities, producing clear phantom images at concentrations as low as 1 mg/ml and enabling successful in vivo tumor imaging in mice following intratumoral injection (95). Incorporating superhydrophilic phosphorylcholine groups adjacent to fluorine atoms in a hyperbranched structure enhanced hydration around fluorine nuclei, resulting in prolonged T2 relaxation times and improved imaging signals (44,95,97). Despite these advantages, the agent showed limited tumor accumulation following intravenous administration due to insufficient targeting capability, indicating the need for further modification to enhance tumor-targeting efficiency.

Hyperbranched polymers with high fluorine content and enhanced molecular mobility have also been explored as effective 19F MRI contrast agents (98). Polymers synthesized via RAFT polymerization of DMAEA (77 mol%) and tFEA (19 mol%), using EGDMA (4 mol%) as a branching agent, resulted in particles ~10 nm in size (98). To improve cytocompatibility, the polymers were chain-extended with PEGMA, producing polymers (P3) with markedly lower cytotoxicity compared with the parent polymer (P1). Functionalization was demonstrated by conjugating mannose to alkyne-terminated polymers (P2) via Huisgen 'click' chemistry (99), yielding a mannose-functionalized polymer (P4). The 19F T2 relaxation time of P1 at 16.4 T was 88 msec at 20 mg/ml, and imaging experiments showed that signal-to-noise ratio increased linearly with concentration, indicating high sensitivity (41). In vivo 19F MRI involved intravenous injection of P1 into mice, with imaging after 2 h revealing prominent 19F signals in the bladder, demonstrating renal excretion and successful imaging within 10 min (37). This work represented the first example of functionalizable hyperbranched fluoropolymers engineered for in vivo 19F MRI with high sensitivity and facile ligand functionalization, offering a promising platform for targeted imaging agents (96). Nevertheless, further investigation into the in vivo efficacy and specificity of the cell-targeted polymers is needed.

19F MRI contrast agents hold significant potential for noninvasive imaging due to the favorable nuclear properties of fluorine and absence of background signals in biological tissues (100). FCy7-NO2, a novel dual-mode near-infrared fluorescence and 19F MRI probe, was designed to image tumor hypoxia by detecting nitroreductase (NTR) activity. FCy7-NO2 incorporates nitro groups as NTR recognition units and fluorine atoms for 19F NMR detection. Upon enzymatic reduction to FCy7-NH2 by NTR, the probe exhibits enhanced fluorescence and significant 19F chemical shift changes. In vitro, FCy7-NO2 showed a sevenfold increase in fluorescence intensity after 1 h incubation with NTR (0.5 μg ml⁻¹) and a 19F NMR chemical shift change from −118.6 ppm to −123.8 ppm. In hypoxic A549 cells, fluorescence intensity increased proportionally with NTR levels, and 19F MRI quantified intracellular NTR concentrations. In vivo, mice with orthotopic lung tumors injected with FCy7-NO2 exhibited a significant increase in fluorescence intensity in the tumor area 24 h post-injection, with the left lung fluorescence intensity 2.9 times higher than the kidney and 7.4 times higher than the liver. Additionally, 19F MRI detected FCy7-NH2 selectively in the tumor region, confirming NTR activity in vivo. This dual-modal probe enables sensitive and selective detection of NTR over a broad concentration range without depth limitations, offering a promising method for noninvasive imaging of tumor hypoxia and understanding tumor evolution (100-103). However, probe aggregation at high concentrations may reduce 19F MRI signal intensity, necessitating further studies to optimize its pharmacokinetics and biodistribution (41,104).

To overcome the limitations of weak thermal polarization in 19F MRI contrast agents hindering low-concentration target detection (105), a study applied CEST principles to achieve a 900-fold signal amplification of a biocompatible fluorinated agent, enabling micromolar detection in a 'multicolor' imaging format (106,107). In vitro experiments confirmed multiplexed detection capability using 19F-GEST MRI, and cell viability assays verified host biocompatibility. Proof-of-concept in vivo studies delivering G4 via inhalation and hosts via intracranial injection in mouse brains demonstrated localized 19F-GEST effects with potential in vivo translatability. This design exploits dynamic exchange in host-guest supramolecular assemblies to amplify the 19F MRI signal of a single fluorinated agent, markedly enhancing applicability for mapping previously undetectable low-abundance targets with micromolar detectability and multiplexed imaging via distinct chemical shifts (24,28,108-112). Limitations include the need for further in vivo optimization, potential chemical shift overlaps at lower magnetic fields, and the preliminary nature of in vivo studies requiring more extensive research.

19F MRI offers significant potential for in vivo molecular and cellular imaging due to its negligible background signal and high specificity. Compared with small-molecule or polymer-based approaches, nanoparticle strategies can load large amounts of fluorine while still permitting functionalization for targeted delivery (25). However, challenges such as MPS clearance and off-target uptake require careful formulation to preserve high signal in desired tissues. Studies have focused on enhancing 19F MRI sensitivity and specificity through innovative nanoparticle designs, addressing issues such as decreased solubility with increased fluorine content, signal attenuation from restricted molecular mobility, and maintaining biocompatibility (108).

Activatable 19F MRI nanoprobes for caspase-1 activity sensing were developed, where tandem repeats of substrate peptide sequences improved turn-on responses, enabling in vivo immune response imaging (112-115). Nanocarriers incorporating fluorinated polyelectrolyte Nafion formed 170 nm particles detectable by 19F MRI, optimized for passive tumor targeting and drug delivery (88,116,117). Dual-mode nanoparticles, such as fluorinated mesoporous silica nanoparticles functionalized with fluorosilane or polyfluorosiloxane and grafted with gadolinium chelates, provided both ¹H and 19F MRI capabilities for anatomical and nanoparticle detection, respectively (118,119). Temperature-responsive polymeric nanogels composed of amphiphilic copolymers formed particles (~120 nm) with good 19F MRI sensitivity and non-cytotoxicity (120-122).

To enhance signal, hyperbranched fluoropolymers were synthesized, forming micelles (20-45 nm) where acrylate-based polymers exhibited stronger MRI signals than methacrylate-based ones (123). Perfluorocarbon nanoparticles effectively labeled human islets without compromising viability or glucose responsiveness, detectable by 19F MRI, computed tomography and ultrasound, demonstrating multimodal imaging potential (124). Targeted delivery was improved using αᵥβ₃-integrin-targeted PFC nanoparticles, with intratracheal administration in lung cancer models resulting in higher tumor PFC concentrations than intravenous routes (125). Superhydrophilic zwitterionic fluorinated polymers with high 19F content (19.1 wt%) showed resistance to protein adsorption and produced intense whole-body 19F MRI signals following intravenous injection (39,97).

However, challenges remain, such as the need for high concentrations of fluorinated micelles for MRI visualization of labeled cells, adversely affecting cell viability (126). Achieving higher labeling rates without compromising viability and ensuring nanoparticle stability are significant hurdles. These studies collectively highlight advances in nanoparticle-based 19F MRI contrast agents, emphasizing innovations in design and synthesis that enhance imaging sensitivity and specificity while addressing biocompatibility and signal detection challenges.

To address limitations of current 19F MRI probes, researchers developed novel multifunctional core-shell nanoparticles called fluorine accumulated silica nanoparticle for MRI contrast enhancement (FLAME) (87,111,119). FLAME nanoparticles consist of a micelle core filled with liquid PFCE, containing 20 equivalent fluorine atoms yielding a sharp 19F NMR peak, encapsulated within a robust silica shell for surface modification, improved biocompatibility and in vivo stability (127-129). Transmission electron microscopy revealed FLAME particles with an mean value diameter of 76±9 nm, and 19F NMR spectroscopy confirmed a single PFCE-derived peak at −16.4 ppm. FLAME demonstrated high 19F MRI signal intensity proportional to PFCE concentration (87,111).

FLAME nanoparticles modified with ampicillin (FLAME-Amp) enabled the specific detection of BL-tag proteins at concentrations as low as 30 nM, markedly outperforming small-molecule probes such as F-Amp, which required micromolar protein concentrations. Notably, the T2 relaxation times of FLAME-Amp remained stable even after protein binding, ensuring consistent MRI signals. In vivo, PEGylated FLAME (FLAME-PEG) accumulated in tumor tissue via the enhanced permeability and retention effect, with strong 19F MRI signals observed at the tumor site, confirming effective delivery and retention (119). The design of FLAME nanoparticles effectively combines high fluorine content with maintained molecular mobility and surface modifiability, enhancing sensitivity and potential for targeted imaging applications. This work represents a significant advancement in developing highly sensitive and versatile 19F MRI contrast agents capable of detecting gene expression in living cells and tumors with improved stability and functionality. However, further studies are needed to fully evaluate the long-term biocompatibility and targeted delivery capabilities of FLAME nanoparticles in clinical settings (127-129).

Another promising approach involves self-assembling supramolecular dendrimers for 19F MRI contrast agents (130,131). These amphiphilic dendrimers comprise a hydrophobic alkyl chain and a hydrophilic dendron with multiple negatively charged fluorinated terminals, preventing aggregation via electrostatic repulsion. This design maintains high fluorine nuclei mobility, achieving a high fluorine content (16.7 wt%) with excellent water solubility ≤85 mg/ml. The dendrimer 1c self-assembled into nanomicelles (~25 nm) exhibiting favorable relaxation properties (T1 of 534-593 msec, T2 >190 msec), surpassing common 19F MRI agents. Importantly, dendrimer 1c was non-toxic and capable of encapsulating the near-infrared fluorescence dye DiR and the anticancer drug paclitaxel, enabling multimodal imaging and theranostics for pancreatic cancer. In vivo studies on human pancreatic cancer xenografts in mice demonstrated that PTX-loaded dendrimer markedly inhibited tumor growth Compared with PTX alone, with 19F MRI and NIRF imaging confirming specific tumor localization. This work provides a promising approach for constructing 19F MRI agents and theranostic systems using self-assembling supramolecular dendrimer chemistry. However, the observed toxicity of lower-generation dendrimers highlights the need for careful structural optimization to ensure safety (129,132,133).

A novel dual nanoparticle conjugate (DNC) platform was developed as an aptamer-based 'turn-on' sensor for 19F MRI. The DNC consists of core-shell nanoparticles with a liquid perfluorocarbon core and a mesoporous silica shell (19F-MSNs) of diameter 94±27 nm, providing a robust 19F MR signal, and superparamagnetic iron oxide nanoparticles (SPIONs) of diameter 4.7±0.6 nm serving as magnetic quenchers (134). Due to the strong T2 relaxation enhancement by SPIONs, effective quenching was achieved with only four equivalents of SPIONs relative to 19F-MSNs. The probe functions via target-induced dissociation of DNA aptamers; specifically, the thrombin-binding aptamer was incorporated as a proof-of-concept (DNCThr). Upon incubation with human α-thrombin at concentrations ≤1 μM, the 19F T2 relaxation time increased markedly from a quenched state of 86 msec to 581 msec, indicating successful turn-on of the MR signal. In vivo experiments demonstrated that DNCThr generated a robust 19F MRI 'hot-spot' signal in response to thrombin injected subcutaneously in live mice, with a SNR of 7.3 compared with 3.2 in control injections. The versatility of the platform was further demonstrated by adapting it to sense ATP and kanamycin, showing a similar increase in T2 relaxation times upon target binding.

Overall, advances in nanoparticle-based 19F MRI contrast agents, including FLAME nanoparticles and supramolecular dendrimers, have spurred meaningful progress in imaging sensitivity and specificity. These innovations address key challenges in biocompatibility, stability, and signal detection, bringing 19F MRI closer to practical applications in molecular and cellular imaging (128,135,136). Continued refinement of nanoparticle designs is crucial for translating these technologies into clinical use.

Fluorinated small molecules have emerged as promising 19F MRI contrast agents due to their well-defined chemical structures, enabling precise characterization, reproducible synthesis, and scalable production. While such small-molecule agents are easier to prepare and often clear quickly, they commonly offer fewer 19F atoms per molecule than polymeric or PFC-based systems, limiting their raw signal strength. Nonetheless, the straightforward design and tunable properties of small-molecule probes allow for specific targeting or responsiveness to physiological stimuli, making them valuable tools for disease diagnosis and drug monitoring (39). Their good aqueous solubility and low molecular weight facilitate efficient biological distribution and rapid renal clearance without requiring extensive modification (137-140). Overall, these profiles present a viable alternative to nanoparticle or polymer-based formulations when short circulation times and low accumulation are desirable.

Fluorinated sugars, which mimic natural biomolecules and participate in metabolic processes, are vital metabolic imaging agents for cancer detection and treatment monitoring. The unique properties of 19F, particularly its chemical inertness and small atomic size, allow these compounds to be recognized by biological systems similarly to their non-fluorinated counterparts (25,39,40). Since endogenous 19F is negligible in biological tissues, 19F NMR and MRI can track these molecules in vivo without background interference. A key example is 2-fluoro-2-deoxy-D-glucose (2-FDG), a fluorinated glucose analog (141). Due to its structural similarity to glucose, 2-FDG is taken up by cells via glucose transporters and phosphorylated to form 2-fluoro-2-deoxy-D-glucose-6-phosphate (2-FDG-6-P) but cannot undergo further glycolysis (141). This leads to its accumulation in cells with high glucose uptake, such as cancer cells, providing a basis for imaging tumor metabolism using 19F MRI. Preclinical studies by Kanazawa et al (142) demonstrated the effectiveness of 2-FDG as a metabolic imaging agent. Injecting 2-FDG into tumor-bearing mice, they obtained 19F MR images revealing higher concentrations of 2-FDG and its metabolite 2-fluoro-2-deoxy-D-mannose (2-FDM) within tumor tissues compared with normal tissues, highlighting their potential for non-invasive tumor detection and metabolic assessment. Furthermore, fluorinated sugars such as 2-FDG offer insights into radioactive counterparts used in positron emission tomography (PET), such as 2-[18F]FDG (141). While PET provides high sensitivity, it involves ionizing radiation and limited availability. By contrast, 19F MRI with fluorinated sugars presents a non-radioactive alternative suitable for repeated longitudinal studies, advantageous for ongoing treatment monitoring (37,41,42,143). Beyond detection, fluorinated sugars aid in monitoring therapeutic responses. Changes in their uptake and metabolism can indicate alterations in tumor metabolism post-treatment, providing early signs of efficacy or resistance. Consequently, they serve as valuable tools in personalized medicine, optimizing therapeutic strategies based on the metabolic profiles of individual tumors.

Fluorinated drugs such as 5-FU and fluoxetine contain fluorine atoms crucial to their therapeutic functions, making them suitable for detection and monitoring via 19F MRI (128,140,144). However, their low fluorine content per molecule limits imaging sensitivity (38,145). To address this, bioorthogonal chemistry strategies have been developed. One approach employs azide-containing small molecules incorporated into cellular components through metabolic processes. These azide groups react with fluorinated cyclooctyne probes via click chemistry, increasing the number of 19F nuclei associated with target cells and amplifying imaging sensitivity (146). This enhancement allows successful deep-tissue visualization of metabolic probes, enabling more accurate and detailed imaging results.

5-FU, a chemotherapeutic agent widely used to treat cancers such as colorectal and breast cancers, undergoes complex metabolic pathways in vivo, converting into active metabolites that disrupt DNA and RNA synthesis in rapidly dividing cells (147). Despite sensitivity challenges, researchers have used 19F MRI to map the biodistribution of 5-FU in tumor-bearing animal models, observing its accumulation in tumors and major organs shortly after administration (128,142). Monitoring the in vivo behavior of 5-FU via 19F MRI aids in optimizing dosing regimens and minimizing systemic toxicity by tailoring treatments to individual patient responses (142). Similarly, fluoxetine, a selective serotonin reuptake inhibitor prescribed for depression and anxiety disorders, contains a fluorine atom detectable by 19F MRI. Visualizing its distribution in the brain and peripheral tissues offers insights into its pharmacokinetics and therapeutic mechanisms. Tracking fluoxetine with 19F MRI could reveal patterns of drug uptake, distribution, and clearance, contributing to personalized medicine approaches in neuropsychiatric treatment.

The development of responsive 19F MRI probes has markedly advanced non-invasive imaging of pathological conditions, including cancer and inflammation. By altering their NMR properties in response to biological stimuli, such as pH changes, enzyme activity, or metal ions, these probes provide real-time insights into cellular and molecular processes (148). These responsive probes integrate fluorine atoms into molecular structures that undergo chemical transformations upon encountering target stimuli. The high sensitivity of 19F NMR to electronic environment changes, coupled with the absence of endogenous fluorine in biological systems, enhances imaging specificity and makes 19F MRI ideal for detecting these transformations.

In cancer detection, elevated glycolysis in tumor cells leads to an acidic microenvironment. To map intra-tumoral pH variations, responsive 19F MRI probes with pH-sensitive moieties have been developed. These fluorinated compounds undergo detectable chemical shift changes in 19F MRI, enhancing assessment of tumor progression and therapeutic effectiveness through spatial pH mapping. Enzyme-responsive 19F MRI probes provide additional specificity by targeting enzymes overexpressed in tumors, such as matrix metalloproteinases (114,128,148,149). Probes with cleavable linkers alter their 19F NMR signal upon enzymatic cleavage. Remaining quenched until activated by the target enzyme, these probes produce a detectable 19F MRI signal where enzyme activity occurs (134).

In inflammation, 19F MRI probes are instrumental in tracking immune cell infiltration and activity (81,128,129). Metal ion-sensitive probes responsive to Ca²⁺ or Zn²⁺ fluctuations during inflammatory responses visualize these processes. Fluorinated chelators binding Zn2+ exhibit changes in 19F NMR relaxation properties, enabling detection of inflammation-associated zinc fluxes. Additionally, reactive oxygen species (ROS)-responsive probes containing boronate esters react with hydrogen peroxide, altering the 19F NMR signal and allowing selective imaging of oxidative stress in diseases such as atherosclerosis or neurodegenerative disorders (150-153). Advanced probe designs incorporate dual functionality by combining 19F MRI with optical fluorescence imaging (154). These dual-modal probes offer complementary information: Fluorescence provides high-resolution localization, while 19F MRI offers deep tissue penetration (154). Using techniques such as aggregation-induced emission fluorophores can circumvent quenching in self-assembled polymeric probes. By mapping physiological changes, pH variations, enzyme activity, and metal ion concentrations, responsive 19F MRI probes provide valuable insights into the onset and progression of diseases such as cancer and inflammation (41,42,128,129).

Advances in 19F MRI contrast agents have led to novel compounds with improved imaging capabilities for diverse biomedical applications. CA-sar-TFMA, a trifluorinated bile acid resistant to CGH-mediated deconjugation, was developed for noninvasive assessment of bile acid transport. It showed favorable in vitro and in vivo stability, acted as a potent inhibitor and substrate of apical sodium dependent bile acid transporter (ASBT) and Na+/taurocholate cotransporting polypeptide (NTCP) and accumulated 16.1-fold more in gallbladders of wild-type mice than Asbt-deficient mice, supporting its potential as an MRI probe for bile acid transport (155).

To improve solubility of fluorine tracers, the hyperfluorinated hydrophilic organofluorine ET1084 (~24 wt% 19F) was developed, achieving water solubility at ≥8 M 19F concentration. Phantom studies at 9.4 T demonstrated a linear increase in SNR with concentration, a detection limit of 5 mM, and preliminary safety ≤20 mM (156). Additionally, water-compatible fluorine-rich polymers were synthesized via nucleophilic addition to enhance 19F MRI signals (37,86). Incorporation of PEG linkers increased T2 without compromising high T1 values, improving NMR signals and peak profiles. Phantom imaging showed bright signals, but clinical translation limitations persist (157).

In pulmonary imaging, octafluorocyclobutane (OFCB) was evaluated as an inhalable 19F MRI contrast agent (29). At 0.5 T, human studies showed anatomically consistent lung images with SNRs of 50 in 2D and 20 in 3D modes using breath-hold durations of 20-40 sec, indicating the clinical potential of OFCB despite resolution limitations due to low field strength (158). Moreover, 19F MRI was used to monitor hydrogel scaffold degradation in vivo, offering precise localization and quantitative degradation rates without endogenous signal interference, suggesting utility for implant evaluation, though further validation is needed (159).

Fluorinated mannoheptulose derivatives (19FMH) have been investigated for imaging GLUT-2-expressing cells. Although 19FMHs preferentially accumulated in GLUT-2-rich tissues and showed potential for cell tracking, rapid clearance and low 19F MRI sensitivity presented challenges requiring optimization (160). Collectively, these findings underscore the wide-ranging efforts to design highly sensitive 19F MRI contrast agents, whether through new small molecules, polymers, nanoparticles, or responsive probes, that highlight promising trends and emerging gaps. Future work must continue improving signal intensity, distribution, safety, and targeted specificity to advance 19F MRI toward widespread clinical adoption.

D-glucose has emerged as a promising non-metallic CEST contrast agent for brain tumor imaging due to its natural presence and favorable safety profile (175,176). Exploiting exchangeable protons in its hydroxyl groups, D-glucose functions in Dynamic Glucose-Enhanced (DGE) MRI by allowing saturation and detection through MRI, monitoring transient changes in glucose concentration within tissues (176). Intravenous administration of glucose enables real-time tracking of its accumulation and washout in brain tissues, and DGE MRI has been successfully translated to human studies, allowing visualization of brain tumors with enhanced contrast.

A significant advantage of DGE MRI is its ability to detect disruptions in the BBB, a hallmark of malignant brain tumors (177). Tumor-induced BBB breakdown permits increased extravasation of glucose into the tumor interstitium compared with normal brain tissue. This differential uptake results in heightened CEST signals within tumors, providing valuable diagnostic information about tumor location, size, and permeability, and aiding in the assessment of tumor aggressiveness and therapeutic planning (178-182). However, at 3 T, the DGE signal change is modest (~1%) and susceptible to motion artifacts, necessitating effective motion correction and optimized infusion protocols. Prolonged infusion durations of 3-4 min help mitigate transient side effects without compromising the DGE signal change, enhancing the robustness of glucoCEST imaging. The translation of D-glucose glucoCEST MRI to human studies at 7 T demonstrated feasibility in detecting dynamic signal changes in glioma patients, with variations in signal enhancement correlating with perfusion properties and BBB permeability (183,184).

To overcome limitations of D-glucose, other sugar analogues have been explored. Non-metabolizable analogues such as 2-deoxy-D-glucose (2-DG) and 3-O-methyl-D-glucose (3-OMG) are structurally similar to glucose but not fully metabolized, allowing prolonged imaging windows and investigation of glucose transport and uptake mechanisms within tumors. 2-DG enters cells via glucose transporters and becomes trapped after phosphorylation, while 3-OMG is transported without subsequent metabolism. Studies using these agents demonstrated improved tumor visualization and insights into tumor metabolism (185,186). For instance, 3-OMG showed around twice the CEST contrast enhancement compared with D-glucose in brain tumors, with tumor regions exhibiting enhancement of 2.5-5.0% vs. 1.5-3.5% in contralateral brain, and prolonged signal persistence (187). Additionally, 2-DG and 2-fluoro-2-deoxy-D-glucose generated significant CEST effects ≤30% persisting over an hour in mammary tumors, suggesting potential to replace PET imaging in preclinical studies (170). Comparative analyses of these analogues indicate that the lack of phosphorylation of 3-OMG may extend its imaging window, while the phosphorylation of 2-DG increases retention within tumor tissues, each approach offering advantages that can be tailored to specific clinical goals (186,187).

Other agents such as glucosamine (GlcN) have been investigated as exogenous CEST contrast agents (188). The anomeric equilibrium and mutarotation rate constants of GlcN, crucial for CEST effects, markedly depend on concentration, pH, and buffer conditions; for example, at pH 7.0 and GlcN concentration of 0.5 M, the mutarotation rate constant was 5.0×10⁻⁴ sec⁻¹, reaching 95% equilibrium in 1.7 h (189). Sugar alcohols such as maltitol have also been proposed; in vivo studies showed CEST contrast elevation in glioma regions due to permeable BBBs, while not affecting normal brain tissue (190). Xylose demonstrated higher sensitivity than glucose in CEST and CESL MRI techniques, without markedly affecting blood glucose levels or neural activity, making it a promising agent for studying glucose uptake (191).

Researchers developed Dex1, the smallest clinically available dextran (~1 kDa), as a new CEST MRI contrast agent to assess tumor hemodynamics, hypothesizing that its hydroxyl protons provide detectable CEST signals and its established safety profile facilitates clinical translation (192). In vivo CEST MRI studies on mice with orthotopic GL261 brain tumors revealed that intravenous injection of Dex1 (2 g/kg) resulted in markedly higher CEST contrast enhancement in tumors compared with contralateral brain tissue (∆MTR_ asym1.2ppm= 0.010±0.006 vs. 0.002±0.008) at 20 min post-injection. Consistent with dynamic contrast-enhanced MRI and fluorescence microscopy, these findings demonstrate the potential of Dex1 as a highly translatable CEST MRI contrast agent. Overall, these studies underscore that, despite promising safety profiles, glucose and glucose analogues still face challenges in achieving robust sensitivity, stable signal detection, and consistent quantitative analysis protocols for full clinical potential (175).

CEST MRI is a novel imaging technique that enables in vivo mapping of metabolites by exploiting proton exchange mechanisms between metabolites and water protons. This method provides contrast based on specific molecular environments, allowing the detection of endogenous molecules such as creatine (Cr), phenol, glycine, and urea (193,194). CEST MRI offers insights into metabolic changes associated with various physiological and pathological conditions, including myocardial infarction (MI), enzymatic activities, neurotransmitter distributions and renal function (168-170). Among these agents, creatine-based contrast is particularly valuable for cardiac remodeling studies, whereas phenol- and glycine-based contrasts facilitate the detection of enzymatic and neurotransmitter abnormalities, respectively, highlighting how each endogenous metabolite addresses different clinical needs.

Cr-weighted CEST MRI can map Cr distribution during MI, offering insights into metabolic changes during myocardial remodeling (195); a study investigated dynamic alterations of myocardial Cr during acute MI using this technique (162). A total of seven adult Bama pigs underwent cardiac cine, Cr-weighted CEST, and late gadolinium-enhanced (LGE) T1-weighted imaging on a 3 T scanner at 3 and 14 days post-MI induction. Cardiac structural and functional indices (MM, EDV, ESV, SV and EF) were assessed, with myocardium categorized as infarct, adjacent, or remote regions based on LGE-determined infarct angle. Cr-weighted CEST MRI signals, reflecting creatine changes, were analyzed using a three-pool Lorentzian model. While MM, EDV, and ESV remained stable (P>0.05), SV and EF rose markedly, and the infarct angle decreased. Cr-weighted CEST signals markedly increased from day 3 to day 14 in infarct, adjacent, and whole myocardium regions. These findings highlight a significant correlation between increased myocardial Cr and structural and functional recovery during acute MI, underscoring the potential of CEST MRI in assessing heart remodeling from a metabolic perspective. Limitations such as small sample size and single-slice imaging may restrict the generalizability of results (196).

Beyond cardiac imaging, CEST MRI has been used with other endogenous compounds to explore various physiological and pathological conditions (106). Phenol has been used as a contrast agent for detecting enzymatic activity. Its exchangeable hydroxyl proton resonates at 4.8 ppm from water and can be detected at sub-millimolar concentrations under acidic conditions (197). Upon acid phosphatase (AcP) activity at pH 5.0, non-CEST-detectable phenyl phosphate is converted to CEST-detectable phenol, enabling direct quantification of AcP activity without a secondary probe (198). This phenolCEST biosensor successfully measured AcP activity in enzyme solutions and prostate cell lysates.

Similarly, GlyCEST MRI has been employed to map glycine levels in the murine brain. Studies revealed higher GlyCEST effects in the thalamus compared with the cerebral cortex (P<0.0001), consistent with biochemical assays (196). In 5xFAD mice, a model of Alzheimer's disease, GlyCEST detected decreased glycine concentrations in the cerebral cortex (P<0.05) and thalamus (P<0.0001), highlighting its potential in investigating neuropsychiatric disorders (196,199).

Additionally, urea has been evaluated as a CEST MRI contrast agent to assess renal concentrating capacity. Phantom experiments demonstrated that urea CEST contrast is concentration and pH-dependent, involving both acid- and base-catalyzed exchange. In vivo studies showed that the inner medulla and papilla exhibited higher pre-injection CEST contrast (2.3±1.9%) compared with the cortex (0.15±0.75%, P = 0.011) and outer medulla (0.12±0.58%, P = 0.008) (200-202). Urea infusion increased CEST contrast in these regions by 2.1±1.9%, whereas saline infusion resulted in a decrease (-0.5±2.0%, P = 0.028 vs. urea), indicating that urea CEST can capture spatial variations in renal function. Practical concerns related to thermal drift, diuretic status and precise pH conditions highlight the need for careful experimental design. Collectively, these studies suggest that endogenous CEST MRI contrast agents offer non-invasive imaging opportunities in diverse contexts, though improving sensitivity and quantitative analysis methods is crucial for broader clinical impact (162).

CEST MRI contrast agents use exchangeable protons to enhance MRI signals, enabling functional imaging applications such as pH mapping. Recent developments have focused on designing novel diamagnetic CEST agents with enhanced imaging properties, sensitivity, and specificity (163,203-208). Nonetheless, variations in chemical shifts, exchange rates and in vivo stability highlight the necessity for systematic comparisons of these compounds to identify the most clinically relevant candidates for pH and perfusion imaging.

A total of 14 newly synthesized imidazole-4,5-dicarboxyamides (I45DCs) were developed and evaluated for pH and perfusion imaging applications (209). These aromatic compounds possess large labile proton chemical shifts (≤7.7 ppm from water) due to intramolecular hydrogen bonds and include a second labile proton for ratio-based pH measurements. The I45DCs demonstrated strong CEST contrast across various substitutions, enabling tuning of the measurable pH range by adjusting inflection points in CEST signal ratio vs. pH plots. Notably, the anionic compound I45DC-diGlu exhibited a ring NH proton exchange rate [k(BA)] of 5081 sec⁻¹ at pH 6.5 and provided a detectable pH range of 5.6-7.0. In vitro studies revealed advantages over currently employed triiodobenzenes for tumor and kidney pH mapping due to larger chemical shifts and tunable pH sensitivity, while cell cytotoxicity assays indicated good tolerability (209-211). In vivo evaluation in a unilateral ureter obstruction mouse model showed that I45DC-diGlu effectively detects functional changes and differences in perfusion and pH between obstructed and unobstructed kidneys, highlighting its potential as a CEST MRI contrast agent for renal imaging. Nevertheless, further investigation into the biocompatibility and quantitative reproducibility of these compounds is necessary prior to clinical adoption.

Additionally, the feasibility of using unlabeled aspirin as an activatable theranostic CEST MRI contrast agent for breast cancer detection has been evaluated (212). By exploiting the conversion of aspirin to salicylic acid (SA), which provides CEST contrast due to exchangeable protons at 9.6 ppm, the study demonstrated that aspirin can serve as a noninvasive theranostic agent. CEST MRI following aspirin treatment showed similar SA CEST contrast (~3%) in both high and low COX-1/-2 expressing breast cancer cell lines, while prostaglandin E2 levels decreased by ~50%. In vivo, mice bearing orthotopic tumor xenografts exhibited tumor contrast enhancement of 5-8% at one h post-injection, with the CEST contrast being dose-dependent. This gadolinium-free imaging approach offers therapeutic effects and imaging capability via a widely used drug. A major limitation is that SA CEST MRI contrast remained independent of COX-1/-2 expression levels, indicating metabolism of aspirin prior to tumor accumulation.

Other studies have advanced the development of diamagnetic CEST agents. Salicylic acid analogues (SAAs) (206), anthranilic acid analogs (213), and phenols with tunable exchangeable protons (214) provide significant contrast at frequencies far from the water resonance (4.8-13.5 ppm), enhancing detection sensitivity. Enzyme-responsive agents synthesized for catalyCEST MRI have demonstrated high specificity in evaluating enzyme activity and inhibition both in vitro and in vivo (215-217), using agents that generate both enzyme-responsive and unresponsive CEST signals for concentration-independent measurements (192). Clinically approved iodinated contrast agents such as iohexol and ioversol have been repurposed as CEST agents, displaying good contrast at 7 T and prolonged tumor enhancement, with significant correlation between CT and CEST-MRI images (R=0.70; P<0.01) (106,218,219). Mannitol, known for osmotic BBB opening, exhibited strong CEST contrast at ~0.8 ppm, enabling non-invasive detection of intracranial accumulation (220). Novel agents such as free-base porphyrins and chlorins provided large upfield shifts (−8 to −13.5 ppm) and suitable exchange rates (500-9,000 sec^−¹) for robust detection (221), while citicoline has been explored as a theranostic agent with inherent CEST signals (222,223). Ongoing work aims to reduce the high concentrations occasionally required for detection (197), address small chemical shifts (221) and mitigate potential toxicity issues (224). Collectively, these developments underscore the importance of refining exogenous CEST MRI contrast agents to address stability, sensitivity, and quantitation challenges for future clinical and theranostic applications (106,205,225).

CEST MRI has emerged as a promising metal-free diagnostic imaging technique, enabling the detection of contrast based on endogenous metabolites, peptides and proteins with minimal invasiveness and low toxicity. Recognizing the limitations of existing genetically encoded CEST contrast agents, which often rely on repetitive amino acid sequences and can pose metabolic or stability issues, researchers developed an in silico method to evolve peptide sequences optimized for CEST contrast, hypothesizing that these peptides could be assembled into a de novo biosensor for CEST MRI (226). The authors designed a synthetic gene encoding a recombinant protein, termed superCESTide, by concatenating top-performing peptides identified through in silico optimization. The resultant protein, consisting of 198 amino acids, exhibited a diverse amino acid composition that reduces reliance on any single residue. SuperCESTide was expressed in Escherichia coli and purified using size exclusion chromatography. CEST MRI assessments at 7 T revealed that the magnetization transfer ratio asymmetry (MTR asym) generated by superCESTide reached a maximum of 6% at 3.6 ppm, comparable to protamine sulfate and human protamine. Faster amide proton exchange rates (474 to 902 sec⁻¹) than poly-L-lysine and numerous endogenous proteins contributed to its enhanced contrast. Challenges remain in purifying superCESTide and characterizing structural stability, suggesting further exploration to ensure reliability and quantify agent performance in complex biological environments.

Researchers modified a lysine-containing peptide (K2) with peptide nucleic acid (PNA) bases at the N-terminus to produce a-K2, c-K2, g-K2, and t-K2 (227), introducing primary amine groups suitable for CEST signal generation. Among these, c-K2 exhibited self-assembly into hydrogels and markedly enhanced the mechanical strength of the hydrogel. The c-K2/g-K2 hydrogel displayed improved mechanical responsivity and good water retention (swelling ratio of 28.6%). These PNA-modified peptide hydrogels generated a detectable CEST signal at ~2.5 ppm due to chemical exchange between exchangeable amine protons and water protons (107). Intratumoral injection into tumor-bearing mice confirmed the capability of hydrogel as an implantable CEST-MRI agent detectable in vivo. Despite their potential, further improvements in gelation properties and evaluation of long-term stability are needed to optimize these systems for clinical feasibility.

Protein and peptide-based CEST MRI contrast agents offer a versatile platform for enhancing imaging specificity via exchangeable protons in amino acid side chains (227-229). Poly(propylene fumarate) scaffolds coated with protamine sulfate demonstrated steady protein release over 24 h, indicating potential for MR-guided drug delivery systems (230). An array of 33 prototype polypeptides showed that the CEST effect can be fine-tuned by altering amino acid sequences (231,232). Methods such as QUEST and QUESP quantified exchange rates in agents such as poly-L-lysine, confirming pH dependence with base-catalyzed exchange predominance (233). Human protamine-1, an arginine-rich peptide, was synthesized as a biocompatible MRI reporter gene, demonstrating markedly higher CEST contrast in engineered cells compared with controls (234,235). Its CEST contrast was highly sensitive to pH, phosphorylation state, and nucleic acids, with binding constants determined by plotting molar concentrations vs. CEST contrast (236). Poly-L-glutamate has been used to map cathepsin expression in vivo, exploiting differences in CEST signals between native and cleaved forms (237). Angiopep-2, an artificial peptide that penetrates the BBB, exhibited a CEST effect peaking at 3.2 ppm with optimal saturation power of 5.5 μT, indicating promise for detecting early Alzheimer's disease (238). A nonmetallic contrast agent, GR-4Am-SA, provided distinct CEST signals at 5.0 and 9.5 ppm to track urokinase plasminogen activator activity with an average reaction coordinate of 80±8% (239). Although these recombinant or modified protein systems can generate high CEST signals, their stability, metabolic effect, and reproducibility must be thoroughly addressed. Continued comparative investigations are essential to determine the optimal strategies for clinical use.

A novel furin-mediated self-assembling olsalazine (Olsa) nanoparticle detectable by both CEST MRI and Raman spectroscopy (240) was developed to target furin-overexpressing tumors. Olsa, a DNA-methylation inhibitor, was conjugated to 2-cyano-6-aminobenzothiazole (CBT) and the furin-specific peptide substrate RVRR to create cell-permeable Olsa-RVRR. Intracellular furin cleaves RVRR, exposing a cysteine residue that reacts with the CBT moiety to produce hydrophobic oligomers, which self-assemble into nanoparticles exhibiting a distinct Raman scattering peak at 1,168 cm⁻¹. In vivo, SCID mice bearing HCT116 xenografts injected with Olsa-RVRR exhibited significant Raman signals in tumors 2 h post-injection, with a 91.7% correct classification rate via support vector machine analysis. This approach offers a potential tool for high-resolution image-guided surgery in furin-overexpressing tumors, though clinical validation is needed to ensure broad specificity.

CEST MRI enables measurement of extracellular pH (pHe) in tumor microenvironments but requires high concentrations of small-molecule contrast agents due to inherent insensitivity (241). To overcome this, nanoscale polymeric CEST agents have been developed to boost CEST sensitivity by increasing the number of exchangeable protons per particle (107,242,243). After optimizing experimental conditions, one study found that a polymer agent enabled acid CEST MRI at concentrations 125-fold lower than a comparable monomer agent, though pH measurements exhibited some concentration dependence (242). In vivo acidoCEST MRI in a xenograft MDA-MB-231 mammary carcinoma model yielded tumor pHe measurements of 6.33±0.12 with iopamidol, 6.70±0.15 with the monomer agent, and 6.85±0.15 with the polymer agent, possibly reflecting differing dosing requirements and complex interactions within the tumor environment. While nanoscale systems can substantially enhance sensitivity, factors such as clearance pathway, potential toxicity, and quantitative accuracy require further optimization.

Nanoparticle-based CEST MRI contrast agents have also been engineered to exploit ionizable tertiary amines (243), carbon dots (244,245) and liposomal formulations (219,222) to enhance imaging contrast in acidic or otherwise specialized physiological conditions. Salicylic acid-conjugated poly(amidoamine) dendrimers produce strong CEST contrast with adjustable proton exchange rates and have shown promise in glioblastoma imaging (246-248). Dual-mode nanoparticles encapsulating perfluoropentane and salicylic acid in hematoporphyrin-poly(lactic acid) polymers have reportedly improved tumor characterization in vitro and in vivo (249). Liposome-based mucus-penetrating particles laden with barbituric acid yielded prolonged vaginal imaging (250). These studies exemplify how nanoparticle strategies can bolster CEST contrast, yet further scrutiny of agent stability and quantitative reproducibility is necessary prior to potential clinical translation.

CEST MRI contrast agents are metal-free alternatives to gadolinium-based agents, providing molecular-level information on key metabolic processes. Using a supramolecular strategy, Pemetrexed was transformed into a molecular hydrogelator with inherent CEST MRI signals; under physiological conditions, it forms filamentous assemblies, creating theranostic hydrogels suitable for injectable delivery and direct monitoring of drug distribution in a mouse glioma model (251). Similarly, paracetamol and acetanilide derivatives have shown significant diamagnetic CEST contrast only when forming intermolecular hydrogen-bonded networks, with paracetamol reaching 12% contrast at 15 mM under physiological conditions (252). A hydrazone-dependent CEST effect (Hydrazo-CEST) derived from N-amino anthranilic acid undergoes a turn-on response upon hydrazone formation with aldehydes, providing an avenue for MRI detection of bioactive aldehydes (253). Meanwhile, N-aryl amides with favorable chemical shifts (4.6-5.8 ppm) allow label-free detection of N-aryl amide drug metabolism (254). A novel 2-HYNIC-based agent for sensing aromatic aldehydes successfully mapped pyridoxal 5′-phosphate in vitro and in vivo in lung cancer models (255). While these supramolecular and label-free strategies expand the range of diamagnetic CEST MRI contrast agents and offer new possibilities for drug delivery monitoring and biomarker detection, further head-to-head comparisons within the broader agent landscape remain essential. Future endeavors should pursue robust validation of safety, improve sensitivity, and address quantitative normalization to enhance the translational potential of these new constructs (106,170,205,225).

Hyperpolarized ¹³C MRI contrast agents enable non-invasive metabolic imaging by markedly enhancing the signal intensity of ¹³C-labeled metabolites, providing valuable insights into physiological and pathological processes, particularly in cancer (321,324-326). Despite their potential, clinical adoption is hindered by the requirement for specialized equipment to excite and detect ¹³C nuclei with MRI gradients, as well as rapid signal decay and operational complexity. Parahydrogen-induced polarization (PHIP) has emerged as a promising alternative to d-DNP for generating hyperpolarized ¹³C-labeled compounds, offering simpler methods for preclinical in vivo MRI (316,318,327,328). Studies have demonstrated the application of PHIP in producing hyperpolarized agents and enhancing metabolic imaging (318,327,328).

One study demonstrated that hyperpolarized allyl [1-¹³C] pyruvate can be detected via stimulated emission of radiation using ¹³C Radiofrequency Amplification by Stimulated Emission of Radiation (¹³C RASER), eliminating the need for RF excitation and synchronization (325). Hyperpolarized allyl [1-¹³C]pyruvate was produced by pairwise addition of parahydrogen to a pyruvate precursor, achieving ¹³C polarization of 4%. RASER signals were detected with a commercial inductive detector at sample concentrations as low as 0.125 M. The PHIP process yielded a mixture of ketone and hemiketal forms, separated by 10 ppm in ¹³C NMR spectra, modeling the metabolic conversion of pyruvate to lactate. Selective, background-free detection was possible when the emission threshold was exceeded only for one species. While this approach could enhance accessibility of metabolic imaging on conventional MRI systems, it also introduced non-physiological concentrations and the use of CD3OD solvent.

Another study developed an automated PHIP-based polarizer capable of producing purified, highly concentrated solutions of hyperpolarized [1-¹³C]fumarate with 13-20% polarization, at physiological pH and in volumes ≤3 ml (326,329). The PHIP process matched d-DNP in polarization levels but reduced preparation time to 10 min compared with 90 min and lowered operational complexity. Cytotoxicity studies confirmed nontoxicity after purification and in vivo experiments showed the use of hyperpolarized fumarate as a perfusion agent and its metabolic conversion to malate in necrotic tumor regions. However, further optimization is needed to reduce residual catalyst contamination for clinical applications and differences in ¹³C relaxation times between PHIP- and d-DNP-prepared samples require further investigation. Comparisons between HP [1-¹³C]pyruvate and [1-¹³C] fumarate highlight how each agent probes a distinct aspect of tumor metabolism, glycolysis vs. necrosis, emphasizing the broader strategy of selecting specific substrates aligned with the biological hallmark of interest.

Additional applications of PHIP include the rapid synthesis of 1-¹³C-pyruvate-d₃, which revealed increased metabolic conversion to lactate and alanine in tumor xenografts (327,328,330). PHIP of 1-¹³C-phospholactate enabled in vivo delivery of a hyperpolarized agent converting to 1-¹³C-lactate, showing uptake in multiple organs and tumors (331). Beyond PHIP, hyperpolarized [1-¹³C]α-ketoglutarate monitored mutant IDH1 activity in glioblastoma, detecting hyperpolarized [1-¹³C]2-hydroxyglutarate exclusively in mutant tumors (332). Hyperpolarized [5-¹³C]glutamine revealed elevated glutamine metabolism in prostate cancer cells and significant metabolic reductions upon treatment, suggesting its potential as a biomarker for therapeutic response (333).

Advances in imaging tumor extracellular pH were achieved using hyperpolarized 1,2-glycerol carbonate, producing HP H¹³CO₃⁻ upon hydrolysis and enabling accurate pHₑ measurements in prostate tumors (334). Similarly, ¹³C-labeled zymonic acid, with a chemical shift change of ≤3.0 ppm per pH unit, allowed reliable extracellular pH mapping in rat kidneys and tumors (335). High-resolution ¹³C NMR spectroscopy at 48.7 mT using hyperpolarized succinate-1-¹³C-2,3-d₂ achieved narrow linewidths (~3 Hz), enhancing sensitivity for monitoring hyperpolarized agent uptake and metabolism (336). Hyperpolarized perfusion imaging with bis-1,1-(hydroxymethyl)-[1-¹³C]cyclopropane-d₈ exhibited distinct perfusion characteristics in glioblastoma, correlating with gadolinium-based MRI and vascular staining (337,338). Simultaneous imaging of multiple ¹³C-labeled agents assessed vascular permeability and perfusion, showing elevated parameters in prostate tumors (339).

Ongoing challenges that require further attention include rapid HP ¹³C signal decay, polarization transfer efficiency, the possibility of residual catalyst contamination with PHIP-based approaches, relaxation time differences between PHIP and d-DNP and complexities in achieving quantitative measurements. These challenges highlight the need for future research into more robust polarization processes, improved agent stability and standardized protocols that improved capture quantitative data from hyperpolarized signals in clinical settings.

Hyperpolarized carbon MRI contrast agents have markedly advanced cardiovascular imaging by enhancing the sensitivity of metabolic and structural assessments in myocardial tissue (147,152,316,318). The conversion of hyperpolarized [1,4-¹³C₂]fumarate to [1,4-¹³C₂] malate serves as a specific probe for cardiomyocyte necrosis post-myocardial infarction (MI) (323,340). In vivo studies demonstrated an 82-times increase in malate production one day after infarct and a sustained 31-times increase after one week Compared with controls, indicating sensitivity to cellular energy status when adenosine triphosphate levels deplete by over 50% (323). Compared with other hyperpolarized agents such as [1-¹³C]acetate, which focuses on oxidative metabolism, [1,4-¹³C₂]fumarate highlights necrosis more directly. Selecting a particular agent thus depends on the specific metabolic process under investigation.

For fast angiography, the SAMBADENA technique enables rapid production of hyperpolarized ¹³C tracers within the MRI system, visualizing major vessels such as the vena cava and aorta in mice, and offering a simple, low-cost alternative to traditional methods (341). Contrast-enhanced magnetic resonance angiography using hyperpolarized ¹³C-enriched compounds has achieved remarkable SNRs (~500) in cardiac regions and preserved magnetization across multiple image acquisitions using flip-back techniques (342).

In metabolic imaging, hyperpolarized ¹³C-labeled glucose analogs have shown myocardial uptake and time-dependent organ distribution in live rats, suggesting applications where radiation-free imaging is preferred over FDG-PET (343). Administration of hyperpolarized [1-¹³C]acetate has been used for studying myocardial metabolism, achieving a liquid-state polarization of 14.2% and an effective in vivo T1 of 17.6±1.7 sec, although requiring large doses (344). For interventional MRI, hyperpolarized ¹³C contrast agents in catheter lumens have provided high SNR (~80) for passive catheter tracking, enhancing real-time visualization (345). Although these methods show promise, ongoing work must address polarization longevity and agent selection to achieve routine clinical use.

Hyperpolarized carbon MRI contrast agents markedly enhance sensitivity for carbon-containing metabolites, facilitating detailed investigations of metabolic pathways (152,316,324,346). The sodium iodide with active background rejection (SABRE) technique extends hyperpolarization of ¹³C nuclei at natural abundance to various α-ketocarboxylates beyond pyruvate, including 2-oxobutyrate (P¹³C=25%), oxaloacetate (P¹³C=11%), α-ketoglutarate (P¹³C=13%), phenylpyruvate (P¹³C=2%) and phenylglyoxylate (P¹³C=2%). While these SABRE-based approaches broaden the range of substrates that can be hyperpolarized without chemical modification, the polarization levels often remain lower than those achieved by d-DNP, underscoring a research gap in optimizing SABRE transfer efficiency for clinical use. Temperature-dependent studies showed that hydride and substrate exchange rates affect polarization levels: Maximum polarization occurred at low temperatures for pyruvate and 2-oxobutyrate, while oxaloacetate benefited from higher temperatures. A theoretical kinetic model fitting the hyperpolarization dynamics provides insights into exchange processes and guides optimization (316,324,346).

By transferring spin order from parahydrogen to the ¹³C nuclei of α-ketocarboxylates via SABRE, NMR signals are enhanced without chemical modification (347). This work broadens SABRE hyperpolarization to more biologically relevant metabolites, but challenges remain for phenyl-substituted substrates due to unfavorable catalyst binding and for compounds, such as oxaloacetate, that undergo decarboxylation under SABRE conditions. Recent advances focus on improving agent design and mechanisms to enhance performance, with proton decoupling techniques such as decoupling pulse sequence (WALTZ-16) eliminating large ¹³C-1H couplings introduced during enzymatic transformations, boosting detection of hyperpolarized [2-¹³C]dihydroxyacetone conversion in vivo (348). Innovations in PHIP methods have led to robust, low-cost parahydrogen generators using liquid nitrogen cooling, achieving parahydrogen fractions over 48% and ¹³C signal enhancements >14,000-fold at 1 T for agents such as sodium [1-¹³C]pyruvate (349).

Integrating PHIP systems into MRI scanners allows virtually continuous production of hyperpolarized agents, such as [1-¹³C]succinate-d₂ and hydroxyethyl-[1-¹³C]propionate-d₃, with polarizations of ~2 and 19%, respectively, every 15 sec, supporting rapid preclinical studies and dynamic metabolic monitoring (350). Deuterated carbon positions in agents such as [1,1,2,2-D₄,2-¹³C]choline chloride extend ¹³C T1 relaxation times and achieve favorable polarization levels (24%), broadening the dissolution DNP MRI agent library (351). Such variations highlight a trend toward designing hyperpolarized compounds with optimized T1, polarization yield, and metabolic relevance. Nonetheless, further refinement is needed to achieve stable, biocompatible agents and consistent polarization levels suitable for clinical translation.

Hyperpolarized carbon MRI contrast agents markedly enhance brain imaging sensitivity, advancing neurodegenerative disease diagnosis and understanding. Cerebral perfusion imaging in rats used hyperpolarized ¹³C-labeled bis-1,1-(hydroxymethyl)-1-¹³C-cyclopropane-D8, enabling calculation of cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) (352). MTT was measured at 2.8±0.8 sec. Although arterial partial-volume effects affected accurate quantification of CBF and CBV, a modified bolus-tracking theory compensated for depolarization effects, improving CBV and MTT accuracy. Hyperpolarized ¹³C-labeled tert-butanol, with T1 values of 46±4 sec in blood (9.4 T) and 43±24 sec in brain tissue, was evaluated as a freely diffusible contrast agent for brain perfusion imaging (353). Dynamic ¹³C imaging in rats achieved high temporal (2-4 sec) and spatial (700 μm in-plane, 2 mm slice thickness) resolution, underscoring its promise for robust, quantitative perfusion measurements.

The novel hyperpolarized molecular probe [1,1,2, 2-D₄,2-¹³C] choline chloride, exhibiting 24% polarization and a T1 of 35 sec at 11.8 T, was introduced for monitoring choline metabolism into acetylcholine in the brain (354). Enzymatic assays confirmed the synthesis of hyperpolarized deuterated-acetylcholine (T1=34 sec at 14.1 T). As various hyperpolarized agents are investigated for brain imaging, comparing PHIP and d-DNP demonstrates a trade-off between faster polarization times and higher polarization levels. Continued progress in balancing sensitivity, agent stability, and clinically feasible hardware is essential to harness the full potential of hyperpolarized carbon MRI in neurodegenerative disease research.

Hyperpolarized carbon MRI contrast agents markedly advance molecular imaging by enabling real-time visualization of metabolic processes with enhanced sensitivity (316,318). An open-source, low-cost PHIP hyperpolarizer produced hyperpolarized ¹³C-succinate with 28% polarization at 30 mM concentration, facilitating ultrafast molecular imaging and multislice ¹³C MRI in mice following tail vein injection (355). Hyperpolarized [¹³C, 15N]urea monitored renal function; glucagon infusion increased renal mean transit time by 14%, demonstrating its utility as a biomarker without affecting sodium distribution, glomerular filtration rate, or oxygen consumption (356).

Advances in DNP formulations include using symmetric anhydrides as precursors for ¹³C-labeled short-chain fatty acids, eliminating the need for glass-forming additives. Employing esterified trityl radicals and lipophilic gadolinium complexes for additive removal further enhances the clinical potential of these MCAs (357). Applications in low-field MRI were demonstrated with hyperpolarized 1-¹³C-succinate-d₂ and 1H-hyperpolarized pyridine, achieving spatial resolutions surpassing micro-PET in 4-8 sec at 47.5 mT, highlighting microscale molecular imaging feasibility at low fields (358). A centrally controlled, automated parahydrogen-based polarizer with in situ detection achieved 20% polarization and a 5,000,000-times signal enhancement at 48 mT, along with an extended T1 relaxation time of 101±7 sec for a ¹³C hyperpolarized contrast agent in water (316,329,359). Additionally, hyperpolarization of barbituric acid derivatives via parahydrogen-induced polarization achieved a 5000-fold ¹³C NMR signal enhancement, indicating their potential as active MRI contrast agents (360).

In situ hyperpolarization within an MRI system at 7 T was demonstrated with 1-¹³C,2,3-2H₂-succinate achieving 11% polarization and an 18,000-fold signal enhancement, mitigating polarization loss during transfer and reducing costs (318,361). Nevertheless, continued improvements are necessary to address stability requirements, minimize polarization loss during reactive steps, and refine imaging techniques that capture transient signal enhancements from strategies such as Parkinson's Disease (362). Low-cost hyperpolarizer designs, improved catalyst removal, and extended T1 retention remain critical for widespread adoption. By tackling these challenges, particularly sensitivity, stability, and robust quantitative analysis, hyperpolarized carbon MRI contrast agents can fulfill their potential for clinical diagnostics and real-time metabolic assessment.