Preclinical Application of CEST MRI to Detect Early and Regional Tumor Response to Local Brain Tumor Treatment.

Treating glioblastoma and monitoring treatment response non-invasively remain challenging. Here, we developed a robust approach using a drug-loaded liposomal hydrogel that is mechanically compatible with the brain, and, simultaneously, we successfully monitored early tumor response using Chemical Exchange Saturation Transfer (CEST) MRI. This CEST-detectable liposomal hydrogel was optimized based on a sustainable drug release and a soft hydrogel for the brain tumor, which is unfavorable for tumor cell proliferation. After injecting the hydrogel next to the tumor, three distinctive CEST contrasts enabled the monitoring of tumor response and drug release longitudinally at 3T. As a result, a continuous tumor volume decrease was observed in the treatment group along with a significant decrease in CEST contrasts relating to the tumor response at 3.5 ppm (Amide Proton Transfer; APT) and at -3.5 ppm (relayed Nuclear Overhauser Effect; rNOE) when compared to the control group (p < 0.05). Interestingly, the molecular change at 3.5 ppm on day 3 (p < 0.05) was found to be prior to the significant decrease in tumor volume on day 5. An APT signal also showed a strong correlation with the number of proliferating cells in the tumors. This demonstrated that APT detected a distinctive decrease in mobile proteins and peptides in tumors before the change in tumor morphology. Moreover, the APT signal showed a regional response to the treatment, associated with proliferating and apoptotic cells, which allowed an in-depth evaluation and prediction of the tumor treatment response. This newly developed liposomal hydrogel allows image-guided brain tumor treatment to address clinical needs using CEST MRI.

Regulatory T cell intravitreal delivery using hyaluronan methylcellulose hydrogel improves therapeutic efficacy in experimental autoimmune uveitis.

Autoimmune uveitis refers to several intraocular inflammation conditions, which are mediated by autoreactive T cells. Regulatory T cells (Tregs) are immunosuppressive cells that have shown potential for resolving various autoimmune diseases, including uveitis. However, poor donor cell dispersion distal to the injection site and plasticity of Treg cells in an inflammatory microenvironment can present obstacles for this immunotherapy. We assessed the use of a physical blend of hyaluronan and methylcellulose (HAMC) as immunoprotective and injectable hydrogel cell delivery system to improve the efficacy of Treg-based therapy in treating experimental autoimmune uveitis (EAU). We demonstrated that the Treg-HAMC blend increased both the survival and stability of Tregs under proinflammatory conditions. Furthermore, we found that the intravitreal HAMC delivery system resulted in a two-fold increase in the number of transferred Tregs in the inflamed eye of EAU mice. Treg-HAMC delivery effectively attenuated ocular inflammation and preserved the visual function of EAU mice. It significantly decreased the number of ocular infiltrates, including the uveitogenic IFN-γ+CD4+ and IL-17+CD4+ T cells. In contrast, intravitreal injection of Treg cells without HAMC only achieved marginal therapeutic effects in EAU. Our findings suggest that HAMC may become a promising delivery vehicle for human uveitis Treg therapy.

Sensitivity schemes for dynamic glucose-enhanced magnetic resonance imaging to detect glucose uptake and clearance in mouse brain at 3 T.

The article from this special issue was previously published in NMR In Biomedicine , Volume 35, Issue 3, 2022. For completeness we are including the title page of the article below. The full text of the article can be read in Issue 35:3 on Wiley Online Library: https://doi.org/10.1002/nbm.4640.

Multiple CEST contrast imaging of nose-to-brain drug delivery using iohexol liposomes at 3T MRI.

Image guided nose-to-brain drug delivery provides a non-invasive way to monitor drug delivered to the brain, and the intranasal administration could increase effective dose via bypassing Blood Brain Barrier (BBB). Here, we investigated the imaging of liposome-based drug delivery to the brain via intranasal administration, in which the liposome could penetrate mucus and could be detected by chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) at 3T field strength. Liposomes were loaded with a computed tomography (CT) contrast agent, iohexol (Ioh-Lipo), which has specific amide protons exchanging at 4.3 ppm of Z-spectrum (or CEST spectrum). Ioh-Lipo generated CEST contrasts of 35.4% at 4.3 ppm, 1.8% at -3.4 ppm and 20.6% at 1.2 ppm in vitro. After intranasal administration, these specific CEST contrasts were observed in both olfactory bulb (OB) and frontal lobe (FL) in the case of 10% polyethylene glycol (PEG) Ioh-Lipo. We observed obvious increases in CEST contrast in OB half an hour after the injection of 10% PEG Ioh-Lipo, with a percentage increase of 62.0% at 4.3 ppm, 10.9% at -3.4 ppm and 25.7% at 1.2 ppm. Interestingly, the CEST map at 4.3 ppm was distinctive from that at -3.4 pm and 1.2 ppm. The highest contrast of 4.3 ppm was at the external plexiform layer (EPL) and the region between left and right OB (LROB), while the CEST contrast at -3.4 ppm had no significant difference among all investigated regions with slightly higher signal in olfactory limbus (OL, between OB and FL) and FL, as validated with histology. While no substantial increase of CEST contrast at 4.3 ppm, -3.4 ppm or 1.2 ppm was observed in OB and FL when 1% PEG Ioh-Lipo was administered. We demonstrated for the first time the feasibility of non-invasively detecting the nose-to-brain delivery of liposomes using CEST MRI. This multiple-contrast approach is necessary to image the specific distribution of iohexol and liposome simultaneously and independently, especially when designing drug carriers for nose-to-brain drug delivery.

Monitor Tumor pHe and Response Longitudinally during Treatment Using CEST MRI-Detectable Alginate Microbeads.

Imaging pHe of the tumor microenvironment has paramount importance for characterizing aggressive, invasive tumors, as well as therapeutic responses. Here, a robust approach to image pH changes in the tumor microenvironment longitudinally and during sodium bicarbonate treatment was reported. The pH-sensing microbeads were designed and prepared based on materials approved for clinical use, i.e., alginate microbead-containing computed tomography (CT) contrast-agent (iopamidol)-loaded liposomes (Iop-lipobeads). This Iop-lipobead prepared using a customized microfluidic device generated a CEST contrast of 10.6% at 4.2 ppm at pH 7.0, which was stable for 20 days in vitro. The CEST contrast decreased by 11.8% when the pH decreased from 7.0 to 6.5 in vitro. Optimized Iop-lipobeads next to tumors showed a significant increase of 19.7 ± 6.1% (p < 0.01) in CEST contrast at 4.2 ppm during the first 3 days of treatment and decreased to 15.2 ± 4.8% when treatment stopped. Notably, percentage changes in Iop-lipobeads were higher than that of amide CEST (11.7% and 9.1%) in tumors during and after treatment. These findings demonstrated that the Iop-lipobead could provide an independent and sensitive assessment of the pHe changes for a noninvasive and longitudinal monitoring of the treatment effects using multiple CEST contrast.

Sensitivity schemes for dynamic glucose-enhanced magnetic resonance imaging to detect glucose uptake and clearance in mouse brain at 3 T.

We investigated three dynamic glucose-enhanced (DGE) MRI methods for sensitively monitoring glucose uptake and clearance in both brain parenchyma and cerebrospinal fluid (CSF) at clinical field strength (3 T). By comparing three sequences, namely, Carr-Purcell-Meiboom-Gill (CPMG), on-resonance variable delay multipulse (onVDMP), and on-resonance spin-lock (onSL), a high-sensitivity DGE MRI scheme with truncated multilinear singular value decomposition (MLSVD) denoising was proposed. The CPMG method showed the highest sensitivity in detecting the parenchymal DGE signal among the three methods, while both onVDMP and onSL were more robust for CSF DGE imaging. Here, onVDMP was applied for CSF imaging, as it displayed the best stability of the DGE results in this study. The truncated MLSVD denoising method was incorporated to further improve the sensitivity. The proposed DGE MRI scheme was examined in mouse brain with 50%/25%/12.5% w/w D-glucose injections. The results showed that this combination could detect DGE signal changes from the brain parenchyma and CSF with as low as a 12.5% w/w D-glucose injection. The proposed DGE MRI schemes could sensitively detect the glucose signal change from brain parenchyma and CSF after D-glucose injection at a clinically relevant concentration, demonstrating high potential for clinical translation.

Deep neural network based CEST and AREX processing: Application in imaging a model of Alzheimer's disease at 3 T.

PURPOSE: To optimize and apply deep neural network based CEST (deepCEST) and apparent exchange dependent-relaxation (deepAREX) for imaging the mouse brain with Alzheimer's disease (AD) at 3T MRI. METHODS: CEST and T1 data of central and anterior brain slices of 10 AD mice and 10 age-matched wild type (WT) mice were acquired at a 3T animal MRI scanner. The networks of deepCEST/deepAREX were optimized and trained on the WT data. The CEST/AREX contrasts of AD and WT mice predicted by the networks were analyzed and further validated by immunohistochemistry. RESULTS: After optimization and training on CEST data of WT mice, deepCEST/deepAREX could rapidly (~1 s) generate precise CEST and AREX results for unseen CEST data of AD mice, indicating the accuracy and generalization of the networks. Significant lower amide weighted (3.5 ppm) signal related to amyloid ß-peptide (Aß) plaque depositions, which was validated by immunohistochemistry results, was detected in both central and anterior brain slices of AD mice compared to WT mice. Decreased magnetization transfer (MT) signal was also found in AD mice especially in the anterior slice. CONCLUSION: DeepCEST/deepAREX could rapidly generate accurate CEST/AREX contrasts in animal study. The well-optimized deepCEST/deepAREX have potential for AD differentiation at 3T MRI.

Relayed nuclear Overhauser enhancement imaging with magnetization transfer contrast suppression at 3 T.

PURPOSE: To develop a pulsed CEST magnetization-transfer method for rapidly acquiring relayed nuclear Overhauser enhancement (rNOE)-weighted images with magnetic transfer contrast (MTC) suppression at clinical field strength (3 T). METHODS: Using a pulsed CEST magnetization-transfer method with low saturation powers (B1 ) and long mixing time (tmix ) to suppress contributions due to strong MTC from solid-like macromolecules, a low B1 also minimized direct water saturation. These MTC contributions were further reduced by subtracting the Z-spectral signals at two or three offsets by assuming that the residual MTC is a linear function between -3.5 ppm and -12.5 ppm. RESULTS: Phantom studies of a lactic acid (Lac) solution mixed with cross-linked bovine serum albumin show that strong MTC interference has a significant impact on the optimum B1 for detecting rNOEs, due to lactate binding. The MTC could be effectively suppressed using a pulse train with a B1 of 0.8 µT, a pulse duration (tp ) of 40 ms, a tmix of 60 ms, and a pulse number (N) of 30, while rNOE signal was well maintained. As a proof of concept, we applied the method in mouse brain with injected hydrogel and a cell-hydrogel phantom. Results showed that rNOE-weighted images could provide good contrast between brain/cell and hydrogel. CONCLUSION: The developed pulsed CEST magnetization-transfer method can achieve MTC suppression while preserving most of the rNOE signal at 3 T, which indicates the potential for translation of this technique to clinical applications related to mobile proteins/lipids change.

Imaging Self-Healing Hydrogels and Chemotherapeutics Using CEST MRI at 3 T.

Imaging hydrogel-based local drug delivery to the brain after tumor resection has implications for refining treatments, especially for brain tumors with poor prognosis and high recurrence rate. Here, we developed a series of self-healing chitosan-dextran (CD)-based hydrogels for drug delivery to the brain. These hydrogels are injectable, self-healing, mechanically compatible, and detectable by chemical exchange saturation transfer magnetic resonance imaging (CEST MRI). CD hydrogels have an inherent CEST contrast at 1.1 ppm, which decreases as the stiffness increases. We further examined the rheological properties and CEST contrast of various chemotherapeutic-loaded CD hydrogels, including gemcitabine (Gem), doxorubicin, and procarbazine. Among these formulations, Gem presented the best compatibility with the rheological (G': 215.3 ± 4.5 Pa) and CEST properties of CD hydrogels. More importantly, the Gem-loaded CD hydrogel generated another CEST readout at 2.2 ppm (11.6 ± 0.1%) for monitoring Gem. This enabled independent and simultaneous imaging of the drug and hydrogel integrity using a clinically relevant 3 T MRI scanner. In addition, the Gem-loaded CD hydrogel exhibited a longitudinal antitumor efficacy of Gem over a week in vitro. Furthermore, the CD hydrogel could be visualized by CEST after brain injection with a contrast of 7.38 ± 2.31%. These natural labels on both the chemotherapeutics and hydrogels demonstrate unique image-guided local drug delivery for brain applications.

Effective Phototheranostics of Brain Tumor Assisted by Near-Infrared-II Light-Responsive Semiconducting Polymer Nanoparticles.

Precise diagnosis and effective treatment of gliomas still remain a huge challenge. Photoacoustic-guided photothermal therapy (PTT) has unique advantages over conventional techniques for brain tumor theranostics, but existing nanoagents for photoacoustic imaging (PAI)-guided PTT are mainly organic small molecules or inorganic nanoparticles, which have the limitations of poor photostability and biocompatibility. Besides, the restricted absorption in the first near-infrared window (NIR-I) of the most existing nanoagents compromises their effectiveness for deep tissue PAI and PTT. We herein develop novel semiconducting polymer nanoparticles (SPNs) that are strongly absorptive in the second NIR window (NIR-II) to alleviate these problems. With the merits of excellent photoacoustic and photothermal performance, high photostability, proper size, and low toxicity, SPNs not only show efficient cellular uptake for PAI and PTT toward U87 glioma cells but also demonstrate effective accumulation in both subcutaneous tumors and brain tumors upon intravenous injection, thereby realizing efficient PAI-guided PTT toward gliomas under NIR-II light irradiation.

Altered d-glucose in brain parenchyma and cerebrospinal fluid of early Alzheimer's disease detected by dynamic glucose-enhanced MRI.

Altered cerebral glucose uptake is one of the hallmarks of Alzheimer's disease (AD). A dynamic glucose-enhanced (DGE) magnetic resonance imaging (MRI) approach was developed to simultaneously monitor d-glucose uptake and clearance in both brain parenchyma and cerebrospinal fluid (CSF). We observed substantially higher uptake in parenchyma of young (6 months) transgenic AD mice compared to age-matched wild-type (WT) mice. Notably lower uptakes were observed in parenchyma and CSF of old (16 months) AD mice. Both young and old AD mice had an obviously slower CSF clearance than age-matched WT mice. This resembles recent reports of the hampered CSF clearance that leads to protein accumulation in the brain. These findings suggest that DGE MRI can identify altered glucose uptake and clearance in young AD mice upon the emergence of amyloid plaques. DGE MRI of brain parenchyma and CSF has potential for early AD stratification, especially at 3T clinical field strength MRI.

CEST MRI detectable liposomal hydrogels for multiparametric monitoring in the brain at 3T.

Adjuvant treatment using local drug delivery is applied in treating glioblastoma multiforme (GBM) after tumor resection. However, there are no non-invasive imaging techniques available for tracking the compositional changes of hydrogel-based drug treatment. Methods: We developed Chemical Exchange Saturation Transfer Magnetic Resonance Imaging (CEST MRI) detectable and injectable liposomal hydrogel to monitor these events in vivo at 3T clinical field. Mechanical attributes of these hydrogels and their in vitro and in vivo CEST imaging properties were systematically studied. Results: The MRI detectable hydrogels were capable of generating multiparametric readouts for monitoring specific components of the hydrogel matrix simultaneously and independently. Herein, we report, for the first time, CEST contrast at -3.4 ppm provides an estimated number of liposomes and CEST contrast at 5 ppm provides an estimated amount of encapsulated drug. CEST contrast decreased by 1.57% at 5 ppm, while the contrast at -3.4 ppm remained constant over 3 d in vivo, demonstrating different release kinetics of these components from the hydrogel matrix. Furthermore, histology analysis confirmed that the CEST contrast at -3.4 ppm was associated with liposome concentrations. Conclusion: This multiparametric CEST imaging of individual compositional changes in liposomal hydrogels, formulated with clinical-grade materials at 3T and described in this study, has the potential to facilitate the refinement of adjuvant treatment for GBM.

Incorporation of an aggregation-induced-emissive tetraphenylethene derivative into cationic gene delivery vehicles manifested the nuclear translocation of uncomplexed DNA.

A fluorophore displaying aggregation-induced emission was introduced at the terminus of branched polyethylenimine (PEI). The formulated polyplex not only demonstrated an improved safety profile and preserved transfection activity but also importantly indicated that the uncomplexed naked DNA rather than the polyplexes translocated into the nucleus.

Highly Efficient Far Red/Near-Infrared Solid Fluorophores: Aggregation-Induced Emission, Intramolecular Charge Transfer, Twisted Molecular Conformation, and Bioimaging Applications.

The development of organic fluorophores with efficient solid-state emissions or aggregated-state emissions in the red to near-infrared region is still challenging. Reported herein are fluorophores having aggregation-induced emission ranging from the orange to far red/near-infrared (FR/NIR) region. The bioimaging performance of the designed fluorophore is shown to have potential as FR/NIR fluorescent probes for biological applications.

Probe Intracellular Trafficking of a Polymeric DNA Delivery Vehicle by Functionalization with an Aggregation-Induced Emissive Tetraphenylethene Derivative.

Characteristic aggregation-induced quenching of π-fluorophores imposed substantial hindrance to their utilization in nanomedicine for insight into microscopic intracellular trafficking of therapeutic payload. To address this obstacle, we attempted to introduce a novel aggregation-induced emission (AIE) fluorophore into the cationic polymer, which was further used for formulation of a gene delivery carrier. Note that the selective restriction of the intramolecular rotation of the AIE fluorophore through its covalent bond to the polymer conduced to immense AIE. Furthermore, DNA payload labeled with the appropriate fluorophore as the Förster resonance energy transfer (FRET) acceptor verified a facile strategy to trace intracellular DNA releasing activity relying on the distance limitation requested by FRET (AIE fluorophore as FRET donor). Moreover, the hydrophobic nature of the AIE fluorophore appeared to promote colloidal stability of the constructed formulation. Together with other chemistry functionalization strategies (including endosome escape), the ultimate formulation exerted dramatic gene transfection efficiency. Hence, this report manifested a first nanomedicine platform combining AIE and FRET for microscopic insight into DNA intracellular trafficking activity.

Aggregation-Induced-Emissive Molecule Incorporated into Polymeric Nanoparticulate as FRET Donor for Observing Doxorubicin Delivery.

Tetraphenylethene (TPE) derivatives characterized with distinct aggregation-induced-emission, attempted to aggregate with doxorubicin (Dox) to formulate the interior compartment of polymeric nanoparticulate, served as fluorescence resonance energy transfer (FRET) donor to promote emission of acceptor Dox. Accordingly, this FRET formulation allowed identification of Dox in complexed form by detecting FRET. Important insight into the Dox releasing can be subsequently explored by extracting complexed Dox (FRET) from the overall Dox via direct single-photon excitation of Dox. Of note, functional catiomers were used to complex with FRET partners for a template formulation, which was verified to induce pH-responsive release in the targeted subcellular compartment. Hence, this well-defined multifunctional system entitles in situ observation of the drug releasing profile and insight on drug delivery journey from the tip of injection vein to the subcellular organelle of the targeted cells.

Dual functionalized amino poly(glycerol methacrylate) with guanidine and Schiff-base linked imidazole for enhanced gene transfection and minimized cytotoxicity.

Efficient transfection activity and minimal toxicity are of crucial importance to the development of gene delivery systems for practical applications. In this work, guanidine and Schiff-base linked imidazole dual functionalized poly(glycerol methacrylate) (IGEP) was firstly synthesized. Subsequent investigations revealed that this new biomedical material is capable of sufficiently binding to plasmid DNA (pDNA) and formulating optimal sized 100 nm nanoparticles with positive ζ potentials of 10-30 mV. Biological evaluations demonstrated the strategic use of guanidine resulting in enhanced cellular uptake and nuclear localization activities by virtue of its favorable affinity to the cellular membrane, and Schiff-base linked imidazole resulting in promoted endosomal escape and DNA cargo releasing behaviors, consequently leading to better transfection efficacy compared to PEI25K in the targeted cells. Another noteworthy fact was guanidine and imidazole minimized cytotoxicity, hence, these advantageous features provided substantial information to construct a safe and efficient gene delivery carrier towards practical development.