Near-Infrared Fluorescence Imaging Contrast Agents for Clinical Research: Limitations and Alternatives

Introduction: Near-infrared fluorescence imaging is a technique that will establish itself in the short term at the international level because it is recognized for its potential to improve the performance of surgical interventions, its moderate investment and operating costs and its portability. Although the technology is now mature, there is currently the problem of the availability of contrast agents to be injected IV. The aim of this methodology article is to propose an alternative solution to the need for contrast agents for clinical research, particularly in oncology. Methodology: They consist of coupling a fluorescent marker in the form of an NHS derivative, such as IR DYE manufactured in compliance with GMP, with therapeutic monoclonal antibodies having marketing authorization for molecular imaging. For a given antibody, the marking procedure must be the subject of a validation file on the final preparation filtered on a sterilizing membrane at 0.22 μm. Once the procedure has been validated, it would be unnecessary to repeat the tests before each clinical research examination. A check of the marking by thin-layer chromatography (TLC) and place it in a sample bank at +4˚C for 1 month of each injected formulation would be sufficient for additional tests if necessary. Conclusion: Molecular near-infrared fluorescence imaging is experiencing development, the process of which could be accelerated by greater availability of clinical contrast agents. Alternative solutions are therefore necessary to promote clinical research in this area. These methods must be shared to make it easier for researchers.

Role of microbubble ultrasound contrast agents in the non-invasive assessment of chronic hepatitis C-related liver disease

Patients who are chronically infected with the hepatitis C virus often develop chronic liver disease and assessment of the severity of liver injury is required prior to considering viral eradication therapy. This article examines the various assessment methods currently available from gold standard liver biopsy to serological markers and imaging. Ultrasound is one of the most widely used imaging modalities in clinical practice and is already a first-line diagnostic tool for liver disease. Microbubble ultrasound contrast agents allow higher resolution images to be obtained and functional assessments of microvascular change to be carried out. The role of these agents in quantifying the state of hepatic injury is discussed as a viable method of determining the stage and grade of liver disease in patients with hepatitis C. Although currently confined to specialist centres, the availability of microbubble contrast-enhanced ultrasound will inevitably increase in the clinical setting.

Nanomaterials incorporated ultrasound contrast agents for cancer theranostics

Nanotechnology provides various nanomaterials with tremendous functionalities for cancer diagnostics and therapeutics.Recently, theranostics has been developed as an alternative strategy for efficient cancer treatment through combination of imaging diagnosis and therapeutic interventions under the guidance of diagnostic results. Ultrasound(US) imaging shows unique advantages with excellent features of real-time imaging, low cost, high safety and portability, making US contrast agents(UCAs)an ideal platform for construction of cancer theranostic agents. This review focuses on the development of nanomaterials incorporated multifunctional UCAs serving as theranostic agents for cancer diagnostics and therapeutics, via conjugation of superparamagnetic iron oxide nanoparticles(SPIOs), Cu S nanoparticles, DNA, si RNA, gold nanoparticles(GNPs), gold nanorods(GNRs), gold nanoshell(GNS), graphene oxides(GOs), polypyrrole(PPy) nanocapsules, Prussian blue(PB) nanoparticles and so on to different types of UCAs. The cancer treatment could be more effectively and accurately carried out under the guidance and monitoring with the help of the achieved theranostic agents. Furthermore, nanomaterials incorporated theranostic agents based on UCAs can be designed and constructed by demand for personalized and accurate treatment of cancer, demonstrating their great potential to address the challenges of cancer heterogeneity and adaptation, which can provide alternative strategies for cancer diagnosis and therapeutics.

Self-assembled superparamagnetic nanoparticles as MRI contrast agents — A review

Recent progress of the preparation and applications of superparamagnetic iron oxide（SPIO） clusters as magnetic resonance imaging（MRI） probes is reviewed with regard to their applications in labeling and tracking cells in vivo, in diagnosis of cardiovascular diseases and tumors, and in drug delivery systems. Magnetic nanoparticles（NPs）, especially SPIO nanoparticles, have long been used as MRI contrast agents and as an advantageous nanoplatform for drug delivery,taking advantage of their unique magnetic properties and ability to function at the molecular and cellular levels. Due to advances in nanotechnology, various means to control SPIO NPs＇ size, composition, magnetization and relaxivity have been developed, as well as ways to usefully modify their surface. Recently, self-assembly of SPIO NP clusters in particulate carriers — such as polymeric micelles, vesicles, liposomes, and layer-by-layer（Lb L） capsules — have been widely studied for application as ultrasensitive MRI probes, owing to their remarkably high spin–spin（T2） relaxivity and convenience for further functionalization.

Combined Pluronic P85- and Ultrasound Contrast Agents-mediated Gene Transfection to HepG2 Cells

This study examined the effect of P85 （a pluronic block copolymer） and microbubble （MB） ultrasound contrast agents under ultrasound irradiation on gene transfection and expression. The pEGFP plasmids that can encode enhanced green fluorescent protein （pEGFP） served as a report gene and were mixed with different concentrations of MB/0.05% （w/v） P85. Then the plasmids were transfected into human hepatoma G2 （HepG2） cells. The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound （US parameters： 1 MHz, 1.0 W/cm2, 20 s, 20% duty cycle）. Twenty-four hours later, the transfection efficiency was assessed by fluorescence microscopy and fluo-rescence activated cell sorting （FACS） analysis. The cell viability was evaluated by Trypan blue exclusion test. The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation. HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein. The expression efficiency reached （22.14±3.06）% and the survival rate was as high as （55.73±3.32）% in the 30% MB plus P85 group. It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.

The combined time-frequency analysis of the acoustic signals backscattered from ultrasonic contrast agents in the evaluation of the blood perfusion

Objective: To analyze the non-periodic, unstable and even chaotic echoes scattered from microbubbles which are extremely sensitive and may easily collapse, fragment or shrink when ultrasound contrast agents are exposed to ultrasound (US) irradiation. Methods: The combined time-frequency analysis was applied to the original signals instead of the traditional Fourier spectral analysis technique. Results: The results obtained from simulation as well as experiment showed that the subharmonic, 2nd harmonic and ultra harmonic of the microbubbles occurred during the oscillation and varied with time. The dependence on the incident ultrasonic amplitude and microbubble parameters were established. Conclusion: The transient echoes backscattered from the ultrasound agent in the evaluation of the blood perfusion can be analyzed thoroughly by the technique of combined-frequency analysis and the time detail of the frequency contents can be revealed.

Advances in magnetic nanoparticle-based magnetic resonance imaging contrast agents

Magnetic resonance imaging(MRI)has revolutionized medical imaging diagnostics with the advantages of non-invasive nature,absence of ionizing radiation,unrestricted penetration depth,high-resolution imaging of soft tissues,organs and blood vessels,and multi-parameter and multi-sequence imaging.Contrast agents(CAs)are crucial for enhancing image quality,detecting molecular-level changes,and providing comprehensive diagnostic information in contrast enhanced MRI.However,the performance of clinical Gd-based CAs represents a limitation to the improvement of MRI sensitivity,specificity,and versatility,thereby impeding the achievement of satisfactory imaging outcomes.In recent years,the development of magnetic nanoparticle-based CAs has emerged as a promising avenue to enhance the capabilities of MRI.Here,we review the advances in magnetic nanoparticle-based MRI CAs,including blood pool CAs,biochemically-targeted CAs,stimulus-responsive CAs,and ultra-high field MRI CAs,as well as the use of CAs for cell labeling and tracking.Additionally,we offer insights into the future prospects and challenges associated with the integration of these nanoparticles into clinical practice.

Intravascular contrast agents in diagnostic applications： Use of red blood cells to improve the lifespan and efficacy of blood pool contrast agents

In medicine, discrimination between pathologies and normal areas is of great importance, and in most cases, such discrimination is made possible by novel imaging technologies. Numerous modalities have been developed to visualize tissue vascularization in cardiovascular diseases or during angiogenic and vasculogenic processes. Here, we report the recent advances in vasculature imaging, providing an overview of the current non-invasive approaches in biomedical diagnostics and potential future strategies for prognostic assessment of vessel diseases, such as aneurysms and coronary artery occlusion leading to myocardial infarction. There are several contrast agents （CAs） available to improve the visibility of specific tissues at the early stage of diseases, allowing for rapid treatment. However, CAs are also hampered by numerous limitations, including rapid diffusion from blood vessels into the interstitial space, toxicity, and low sensitivity. Extravasation from blood vessels leads to a rapid loss of the image. If the contrast medium can fully be confined to the vascular space, high-resolution structural and functional vascular imaging could be obtained. Many scientists have contributed new materials and/or new carrier systems. For example, the use of red blood cells （RBCs） as CA-delivery systems appears to provide a scalable alternative to current procedures that allows adequate vascular imaging. Recognition and removal of CAs from the circulation can be prevented and/or delayed by using RBCs as biomimetic CA-carriers, and this technology should be clinically validated.

Characterization of the interaction of nanobubble ultrasound contrast agents with human blood components

Nanoscale ultrasound contrast agents,or nanobubbles,are being explored in preclinical applications ranging from vascular and cardiac imaging to targeted drug delivery in cancer.These sub-micron particles are approximately 10x smaller than clinically available microbubbles.This allows them to effectively traverse compromised physiological barriers and circulate for extended periods of time.While various aspects of nanobubble behavior have been previously examined,their behavior in human whole blood has not yet been explored.Accordingly,herein we examined,for the first time,the short and long-term effects of blood components on nanobubble acoustic response.We observed differences in the kinetics of backscatter from nanobubble suspensions in whole blood compared to bubbles in phosphate buffered saline(PBS),plasma,or red blood cell solutions(RBCs).Specifically,after introducing nanobubbles to fresh human whole blood,signal enhancement,or the magnitude of nonlinear ultrasound signal,gradually increased by 22.8±13.1%throughout our experiment,with peak intensity reached within 145 s.In contrast,nanobubbles in PBS had a stable signal with negligible change in intensity(1.7±3.2%)over 8 min.Under the same conditions,microbubbles made with the same lipid formulation showed a56.8±6.1%decrease in enhancement in whole blood.Subsequent confocal,fluorescent,and scanning electron microscopy analysis revealed attachment of the nanobubbles to the surface of RBCs,suggesting that direct interactions,or hitchhiking,of nanobubbles on RBCs in the presence of plasma may be a possible mechanism for the observed effects.This phenomenon could be key to extending nanobubble circulation time and has broad implications in drug delivery,where RBC interaction with nanoparticles could be exploited to improve delivery efficiency.

Nanogels as Contrast Agents for Molecular Imaging

Nanogels （NGs） as soft nanosized materials have gained a variety of interests in biomedical fields. The versatile NG scaffolds with 3-dimensional spherical shape, high loading efficiency, tunable surface functionalization, and excellent biocompatibility afford their uses as carrier to load mono- or multi-mode molecular imaging contrast agents （CAs）. This review summarizes the synthesis routes and applications of NGs as CAs for molecular imaging applications including magnetic resonance （MR）, computed tomography （CT）, radionuclide, optical, and dual/mul- ti-modality imaging.

Nanoparticulate X-ray CT contrast agents

X-ray computed tomography(CT) has been widely used as a powerful diagnostic tool in clinics because it can provide high-resolution 3D tomography of the anatomic structure based on the distinctive X-ray absorptions between different tissues. Currently, CT contrast agents are mainly small iodinated molecules, which suffer from drawbacks such as short blood- retention time, nonspecific in vivo biodistribution, and renal toxicity. Utilization of nanoparticles as potential CT contrast agents to overcome the aforementioned issues has advanced rapidly. In this mini review, we introduce current research efforts in the development of nanoparticulate CT contrast agents and discuss the challenges for additional breakthroughs in this field.

Hybrid Dextran-gadolinium Nano-suitcases as High-relaxivity MRI Contrast Agents

Dextran-poly（glycidyl methacrylate） （Dex-PGMA） nano-suitcases were synthesized efficiently via a graft copolymerization induced self-assembly （GISA） approach. On this basis, the Dex-PGMA nano-suitcases were modified with hydrazide, and the attachment of multiple chelated Gd（III） ions to the interior of the nano-suitcases affords nanoscale MRI contrast agents with high relaxivity values. The highly fenestrated dextran shell of the nano-suitcases assures water exchange which readily occurs between the surrounding environment and the Gd（III） ions encapsulated within the hybrid nano-suitcases. The complexation between the hydrophilic hydrazide interior of the nano-suitcases and Gd（III） ions results in an impressive Gd payload at 22.6 wt% in the hybrid nano-suitcases. The longitudinal relaxivity （rl） of the hybrid nano-suitcases is reported as 44.4 L/（mmol-s）, which is 9-14 folds of that of commercial Gd-DTPA agents. In vivo MRI studies demonstrate that the hybrid nano-suitcases accumulated in the lymph node of the rat due to their nanoscale dimensions and displayed strong signals in vivo. The results indicated that the hybrid nano-suitcases provide a promising platform for the diagnosis of lymph node related diseases.

Recent advances on stimuli-responsive macromolecular magnetic resonance imaging (MRI) contrast agents

Magnetic resonance imaging(MRI) has been extensively used in clinical diagnosis and currently over 30% MRI runs are performed in the presence of contrast agents. However, commercially available contrast agents originated from small molecules typically exhibit relatively low relaxivities and insufficient circulation time. Therefore, there is a long pursuit to develop new contrast agents with high relaxivities to discriminate pathological tissues from normal ones. Compared with small molecule MRI contrast agents, the incorporation of small molecule contrast agents into macromolecular scaffolds allows for constructing macromolecular MRI contrast agents, remarkably elevating the relaxivities due in part to increased rotational correlation time(τR). Moreover, if the macromolecular scaffolds are responsive to external stimuli, the MRI signals could be selectively switched on at the desired sites(e.g., pathological tissues), further intensifying the imaging contrast. In this feature article, we outline the recent achievements in the fabrication of stimuli-responsive macromolecular MRI contrast agents. Specifically, macromolecular contrast agents being responsive to acidic p H, redox potentials, and other stimuli including photoirradiation, pathogens, and salt concentration are discussed. These smart contrast agents could affect either longitudinal(T1) or transverse(T2) relaxation times of water protons or other nuclei(e.g.,19 F), exhibiting enhanced signals in pathological tissues yet suppressed signals in normal ones and displaying promising potentials in in vitro and in vivo MRI applications.

Ultrasound contrast agents from microbubbles to biogenic gas vesicles

Microbubbles have been the earliest and most widely used ultrasound contrast agents by virtue of their unique features:such as non-toxicity,intravenous inject-ability,ability to cross the pulmonary capillary bed,and significant enhancement of echo signals for the duration of the examination,resulting in essential preclinical and clinical applications.The use of microbubbles functional-ized with targeting ligands to bind to specific targets in the bloodstream has further enabled ultrasound molecular imaging.Nevertheless,it is very challenging to utilize targeted microbubbles for molecular imaging of extra-vascular targets due to their size.A series of acoustic nanomaterials have been developed for breaking free from this constraint.Especially,biogenic gas vesicles,gas-filled protein nanostructures from microorganisms,were engineered as thefirst biomolecular ultrasound contrast agents,opening the door for more direct visual-ization of cellular and molecular function by ultrasound imaging.The ordered protein shell structure and unique gasfilling mechanism of biogenic gas vesicles endow them with excellent stability and attractive acoustic responses.What’s more,their genetic encodability enables them to act as acoustic reporter genes.This article reviews the upgrading progresses of ultrasound contrast agents from microbubbles to biogenic gas vesicles,and the opportu-nities and challenges for the commercial and clinical translation of the nascentfield of biomolecular ultrasound.

Advances in magnetic resonance imaging contrast agents for glioblastoma-targeting theranostics

Glioblastoma(GBM)is the most aggressive malignant brain tumour,with a median survival of 3 months without treatment and 15 months with treatment.Early GBM diagnosis can significantly improve patient survival due to early treatment and management procedures.Magnetic resonance imaging(MRI)using contrast agents is the preferred method for the preoperative detection of GBM tumours.However,commercially available clinical contrast agents do not accurately distinguish between GBM,surrounding normal tissue and other cancer types due to their limited ability to cross the blood-brain barrier,their low relaxivity and their potential toxicity.New GBM-specific contrast agents are urgently needed to overcome the limitations of current contrast agents.Recent advances in nanotechnology have produced alternative GBM-targeting contrast agents.The surfaces of nanoparticles(NPs)can be modified with multimodal contrast imaging agents and ligands that can specifically enhance the accumulation of NPs at GBM sites.Using advanced imaging technology,multimodal NP-based contrast agents have been used to obtain accurate GBM diagnoses in addition to an increased amount of clinical diagnostic information.NPs can also serve as drug delivery systems for GBM treatments.This review focuses on the research progress for GBMtargeting MRI contrast agents as well as MRI-guided GBM therapy.

Novel nanovectors as liver targeting MRI contrast agents

Accurate diagnosis of hepatocellular carcinoma(HCC) in the early stage is vital for its treatment.Contrast-enhanced dynamic magnetic resonance imaging(MRI) performed in the presence of extracellular contrast agents such as gadolinium chelates is considered as a useful approach for detecting and characterizing focal liver lesions.However,the sensitivity and specificity of conventional MRI contrast agents are far from satisfaction for the detection and characterization of benign and malignant focal liver lesions in the early stage.The novel molecular contrast agents special for liver with relatively longer metabolic time and stable contrast effect in liver tissue are highly desired.The development of nanotechnology provides an unprecedented opportunity for the diagnostic detection rate of HCC and cell-surface receptor-targeted nanotechnology improves the specificity of the detection of focal liver lesions.In order to maximize lesion detection and characterization,novel gadolinium chelates loaded nanovectors including the solid lipid nanoparticles,nanocomplexes and polymeric nanoparticles have been used as biocompatible molecular MRI contrast agent.In this review,the characterization and the advantages/disadvantages of these Gd-loaded novel nanovectors used as molecular MRI contrast agents were discussed.Furthermore,liver target nanovectors aimed at improving the diagnostic accuracy of liver MRI by targeting additional features of focal liver lesions were highlighted.

A PIID-DTBT based semi-conducting polymer dots with broad and strong optical absorption in the visible-light region： Highly effective contrast agents for multiscale and multi-spectral photoacoustic imaging

As a hybrid imaging technique, photoacoustic imaging （PAI） can provide multiscale morphological information of tissues, and the use of multi-spectral PAI （MSPAI） can recover the spatial distribution of chromophores of interest, such as hemoglobin within tissues. Herein, we developed a contrast agent that can very effectively combine multiscale PAI with MSPAI for a more comprehensive characterization of complex biological tissues. Specifically, we developed novel PIID-DTBT based semi-conducting polymer dots （Pdots） that show broad and strong optical absorption in the visible-light region （500-700 nm）. The performances of gold nanoparticles （GNPs） and gold nanorods （GNRs）, which have been verified as excellent photoacoustic contrast agents, were compared with that of the Pdots based on the multiscale PAI system. Both ex vivo and in vivo experiments demonstrated that the Pdots have better photoacoustic conversion efficiency at 532 nm than GNPs and showed similar photoacoustic performance with GNRs at 700 nm at the same mass concentration. Photostability and toxicity tests demonstrated that the Pdots are photostable and biocompatible. More importantly, an in vivo MSPAI experiment indicated that the Pdots have better photoacoustic performance than the blood and therefore the signals can be accurately extracted from the background of vascular-rich tissues. Our work demonstrates the great potential of Pdots as highly effective contrast agents for the precise localization of lesions relative to the blood vessels based on multiscale PAI and MSPAI.

Oral contrast agents lead to underestimation of dose calculation in volumetric-modulated arc therapy planning for pelvic irradiation

Background:The effects of oral contrast agents(OCAs)on dosimetry have not been studied in detail.Therefore,this study aimed to examine the influence of OCAs on dose calculation in volumetric-modulated arc therapy plans for rectal cancer.Methods:From 2008 to 2016,computed tomography(CT)images were obtained from 33 rectal cancer patients administered OCA with or without intravenous contrast agent(ICA)and 14 patients who received no contrast agent.CT numbers of organs at risk were recorded and converted to electronic densities.Volumetric-modulated arc therapy plans were designed before and after the original densities were replaced with non-enhanced densities.Doses to the planned target volume(PTV)and organs at risk were compared between the plans.Results:OCA significantly increased the mean and maximum densities of the bowels,while the effects of ICA on these parameters depended on the blood supply of the organs.With OCA,the actual doses for PTV were significantly higher than planned and doses to the bowel increased significantly although moderately.However,the increase in the volume receiving a high-range doses was substantial the absolute change of intestine volume receiving≥52 Gy:1.46[0.05-3.99,cubic centimeter range:-6.74 to 128.12],the absolute change of colon volume receiving≥50 Gy:0.34[0.01-1.53 cc,range:-0.08 to 3.80 cc].Dose changes due to ICA were insignificant.Pearson correlation showed that dose changes were significantly correlated with a high intestinal volume within or near the PTV(ρ>0.5,P<0.05)and with the density of enhanced intestine(ρ>0.3,P<0.05).Conclusions:Contrast agents applied in simulation cause underestimation of doses in actual treatment.The overdose due to ICA was slight,while that due to OCA was moderate.The bowel volume receiving≥50Gy was dramatically increased when OCA within the bowel was absent.Physicians should be aware of these issues if the original plan is barely within clinical tolerance or if a considerable volume of enhanced intestine is within or near the PTV.

Gadolinium-based contrast agents built of DO3A-pyridine scaffold:Precisely tuning carboxylate group for enhanced magnetic resonance imaging

It is greatly desired to develop novel gadolinium-based contrast agents(GBCAs)as improved platforms for magnetic resonance imaging(MRI).Herein,we report the syntheses of a series of nonionic cyclenbased GBCAs by precisely tuning carboxylate group on DO3A-pyridine scaffold.[Gd-DO3A-4cp]is isolated which adopts an octadentate coordination mode with a free carboxylate group at 4-position of pyridine.It shows the r_(1)relaxivity of 5.8(mmol/L)^(-1)s^(-1)(3 T,25℃),which is 75%higher than 3.3(mmol/L)^(-1)s^(-1)of the clinic used[Gd-DOTA].The possible mechanisms behind the enhanced relaxivity are investigated and proposed by structure-property relationship studies.After validation of low cytotoxicity and considerable kinetic inertness,in-vivo studies are further examined,demonstrating its good MRI performance,biodistribution as well as the way of excretion.

Tumor microenvironment-responsive contrast agents for specific cancer imaging:a narrative review

Molecular imaging is of great significance for early diagnosis and timely treatment of cancer and disease,as well as basic medical and biological research.As personalized cancer treatment has become increasingly popular,the demand for more advanced imaging technologies has also significantly increased.Taking advantage of differences between the tumor microenvironment and normal tissue cells,tumor microenvironment-responsive or"turn-on"contrast agents have a higher signal-to-noise ratio and lower background interference compared with"turn-off"probes,which can remarkably improve the performance of tumor diagnostics.Thus,tumor microenvironment-responsive contrast agents can not only detect changes in the tumor microenvironment,but also have important significance for tumor diagnosis,prediction of invasion potential,evaluation of treatment effectiveness,planning of therapeutic regimens,and tumor prognosis.Herein,this review focuses on recent research progress of tumor microenvironment-responsive intelligent probes,and highlights future research directions of tumor microenvironment-responsive contrast agents for precision diagnostics.