Nanoparticles: Untying the Gordian Knot in Conventional Computed Tomography Imaging

Abstract

Open AccessCCS ChemistryMINI REVIEW1 Jun 2021Nanoparticles: Untying the Gordian Knot in Conventional Computed Tomography Imaging Zhen Sun, Weihua Chen, Wenbo Sun, Bin Yu, Qianqian Zhang and Lehui Lu Zhen Sun State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Science and Technology of China, Changchun 130022 University of Science and Technology of China, Hefei, Anhui 230026 Google Scholar More articles by this author , Weihua Chen State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Science and Technology of China, Changchun 130022 University of Science and Technology of China, Hefei, Anhui 230026 Google Scholar More articles by this author , Wenbo Sun College of Materials Science and Engineering, College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Instrumental Analysis Center of Qingdao University, Qingdao University, Qingdao 266071 Google Scholar More articles by this author , Bin Yu State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Science and Technology of China, Changchun 130022 Google Scholar More articles by this author , Qianqian Zhang State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Science and Technology of China, Changchun 130022 University of Science and Technology of China, Hefei, Anhui 230026 Google Scholar More articles by this author and Lehui Lu *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Science and Technology of China, Changchun 130022 University of Science and Technology of China, Hefei, Anhui 230026 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100807 SectionsAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail X-ray computed tomography (CT) imaging plays an essential role in disease diagnosis due to its noninvasive, painless mode and superior penetration depth. However, the resolution of the soft tissue and minor lesions remains limited. And the disadvantages of conventional contrast agents, such as their inefficient targeting capability, poor biocompatibility, and short circulation times, are considered intractable in clinical use. To overcome these “Gordian knots,” nanoparticles (NPs) for CT imaging have been developed. The advantages of NPs are their exceptionally high sensitivity to X-ray, better imaging performance in vivos and even therapeutic effects. In particular, based on various designs, NP contrast agents composed of different materials integrate multiple imaging modalities, make up for the inadequacy of a single imaging type, and thus provide more accurate information for diagnosis. This review focuses on NPs for X-ray CT imaging and their multifunctional designs. Some perspectives of crucial problems and prospective challenges are also discussed. Download figure Download PowerPoint Introduction The outbreak of the novel coronavirus in 2019 (COVID-19) has posed grave threats to the health of people around the world, and X-ray computed tomography (CT) imaging has played a significant role in clinical diagnosis by virtue of its immediacy and validity.1–3 Since X-ray CT emerged in the 1970s, it has been widely used in numerous applications such as mining,4 agriculture,5 food,6 and especially disease diagnosis,7,8 on account of its ability to image structural and morphological properties in three dimensions. Owing to the distinctive degree to which X-rays of different components can be attenuated, X-ray CT can readily distinguish the electron-dense bones and the permeable surrounding tissues under X-ray irradiation, hence providing high accuracy in the imaging of bone injuries.9,10 However, it is of limited value in assessing soft tissues, such as fat, muscle, and tumors, resulting in the administration of contrast agents in CT imaging, which is called contrast-enhanced CT.11 Since the uptake of contrast agents in different soft tissues, where they exhibit excellent absorption of X-ray, is highly variable, it helps to enhance the contrast for discriminating soft tissues. Up to now, organic iodinated molecules have been the major contrast agents used for contrast-enhanced CT scans,12 and they have been frequently challenged for the following limitations: (1) short circulation time resulting from their small molecular weight, which makes them rapidly metabolized by the kidneys13; (2) poor biocompatibility leading to side effects such as nausea, fever, vomiting, and so forth11; (3) intravenous injection of the untargeted iodinated molecules that makes the dosage and efficiency hard to determine precisely. In addition, CT imaging still cannot be mentioned in the same breath with other imaging modalities, for example, magnetic resonance imaging (MRI) with its the unique ability to image soft tissue14 and positron emission tomography (PET) with its sensitive and quantitative detection of different diseases.15 A promising trend in imaging science and technology is the combination of multiple imaging modalities. The development of nanoparticles (NPs) provides major new insight in untying the “Gordian knots” of conventional CT contrast agents.16,17 Research has shown the application potential in vivo for the following reasons: (1) prolonged circulation time and metabolizable capacities due to the appropriate size, accompanied by excellent biocompatibility18; (2) enhanced permeability and retention (EPR) effects that confer the ability of passive targeting19; (3) the modifiable surface enables the integration of multiple functions into a single particle.20 Besides, the photoelectron effect dominates the contribution to X-ray attenuation, which is related to the atomic number and K-shell electron binding energy.21 In other words, the chemical elements with higher atomic numbers and whose K-edges are in the range of the X-ray spectrum (57–69 KeV) will exhibit outstanding performance in CT imaging, which also guides the novel options in the materials of NPs contrast agents.22 Herein, we review the recent developments in iodine-based NPs and the innovative elements for CT contrast agents. The examples of the X-ray attenuation of NPs contrast agents based on different elements are listed in Table 1. In addition, as an emerging material, the potential of perovskite for CT imaging has been introduced due to its high sensitivity to X-ray. We also highlight the multimodal imaging which can make up for the gaps between CT and other imaging, and thus synergistically improve the precision and accuracy of diagnosis (Figure 1). Table 1 | The X-ray Attenuation Capacity of NPs Contrast Agents Based on Different Elements Element Formation of Contrast Agents X-ray Attenuation Capacity References Iodine Iodinated polymer NPs 34.4 HU·mL/mg [23] Gold Polyethyleneimine-stabilized Au NPs 21.18 HU·mL/mg [24] Bismuth Bi2S3 nanocrystals in polypeptide shell 126.8 HU·mL/mg [25] Ytterbium Upconversion NPs coated with MnO2, chlorine 6 and atovaquone 24.5 HU·mL/mg [26] Tantalum 2D ultrathin tantalum carbide nanosheets 155.284 HU·mL/mg [27] Sliver Micelles include Ag2S NPs, iron oxide NPs and fluorophore 34.7 HU·mL/mg [28] Copper Ultrasmall Cu2−xSe NPs 117.54 HU·mL/mg [29] Platinum Hollow Pt nanoframes 27.65 HU·mL/mg [30] Figure 1 | The various metal-based NPs for CT imaging and the multimodal imaging benefit from the development of NPs. Reprinted with permission from ref 31. Copyright 2020 WILEY-VCH; ref 32. Copyright 2016 WILEY-VCH; ref 33. Copyright 2019 WILEY-VCH; ref 34. Copyright 2018 American Chemical Society; ref 35. Copyright 2019 American Chemical Society; ref 36. Copyright 2019 WILEY-VCH; ref 37. Copyright 2018 Ivyspring; ref 38. Copyright 2020 American Chemical Society. SPECT, single-photon emission computed tomography; PTT, photothermal therapy. Download figure Download PowerPoint Contrast Agents of Various Nanomaterials Iodine-based contrast agents Currently, the number of CT scan examinations is approximately 300 million per year, of which 40% are contrast-enhanced.39 Since 1923, when sodium iodide was first used for delineating the bladder,40 iodine-based contrast agents have been developed following the constant evolution of CT imaging. Clinically used contrast agents such as iopromide,41 iohexol,42 and iodixanol43 make up the vast majority on the market. Though the optimized design makes it low in toxicity and cost-effective, the intravenous injection of such small molecules also leads to low enrichment and rapid renal excretion, which has been identified to be related to acute kidney injury.44 The emergence of nanomaterials provides new opportunities to develop this diagnostic modality. Increasingly people focus on the iodine-rich NPs, for example, dendrimers, block copolymer micelles, and emulsions, to overcome the above disadvantages.45–49 Dendrimers with branched, layered architectures are used extensively in biomedical applications. They are chemically controllable on which to graft molecules with high loading content.50 Blum’s group49 developed a novel iodine-rich dendrimer using activity-based probes (ABP) methodology. The iodine tags were used as iopanoic derivatives to react with the abundant amino groups of polyamidoamine (PAMAM), which enabled great loading efficiency (Figure 2a). There was also a protein recognition sequence at the other end of the linker, resulting in better targeting ability. The prolonged circulation time and increase in the concentration of iodine reduced the concentration required for signal detection by 15 times. Figure 2 | (a) Structures of an iodine-rich dendrimer, including six iodine tags, fluoroprobe Cy5, and targeting moiety, followed by in vivo CT imaging. (b) Schematic illustration of self-assembled nanopolymersomes with unique features for enhanced CT imaging. (c) Energy-dispersive spectrometer elements mapping of heterojunction nanocrystals and the in vivo imaging effect. Reprinted with permission from ref 49. Copyright 2018 American Chemical Society; ref 46. Copyright 2017 WILEY-VCH; ref 57. Copyright 2019 American Chemical Society. cRGD/IPs, iodine-rich nanopolymersomes decorated with cRGDfK cyclic peptide; CSA, copper selenide-gold. Download figure Download PowerPoint Complex synthesis and high viscosity are the problems faced by dendrimers. In light of this, Zhong and co-workers46 developed biodegradable nanopolymersomes with high iodine content (Figure 2b). The newly designed iodine-functionalized trimethylene carbonate (IC) monomer formed poly(ethylene glycol)-b-poly(iodine trimethylene carbonate (PEG-b-PIC) using ring-open polymerization, and this diblock copolymer was prone to growth into nanopolymersomes by self-assemble, with a hydrodynamic size of ≈100 nm. This system exhibited excellent performance in CT imaging on account of its stable, biodegradable structure with low viscosity and iso-osmolality. The ultralong circulation time is also suitable for blood pool imaging.46 Gold-based NPs In consideration of the mechanism of CT imaging mentioned earlier, gold (Au) NPs have been extensively researched as contrast agents due to the high atomic number (ZAu = 79) and the X-ray attenuation properties (absorption edge kAu = 81 KeV). The size and shape of Au NPs are controllable, and the modification of the surface makes it simple to increase the stability and endow other functions, tumor targeting for example.51 Besides, the strong localized surface plasmon resonance (LSPR) and photothermal conversion efficiency of Au NPs52,53 leading to the integration of therapy and diagnosis with further prospects for application. More recently, a kind of gold bipyramid (Au-BP) with high yield for CT imaging has been developed by Zhang’s group.54 The excellent stability and biocompatibility of Au-BPs were expressed after PEGylated. In addition to the delicate control of size and shape, it was observed that the CT signal was significantly enhanced compared with iopromide, and the absorption was tuned from 740 nm to 960 nm for photothermal therapy. For inorganic metal NPs, construct heterojunction structures are the most expedient means to improve optical performance due to the interlacing of energy levels between heterogeneous components.55,56 These were also used to enhance the overall absorption of X-ray by Li’s group.57 They designed heterogeneous copper selenide-gold (CSA) nanomaterials with dumbbell shape for use against tumors (Figure 2c). The overall X-ray attenuation coefficients (μ) and effective atomic numbers ( Z ¯ ) of CuSe2.26Au1.75 can be estimated by eqs. 1 and 2, respectively: μ CuSe 2.26 Au 1.75 = 0.1 1 μ Cu + 0.3 μ Se + 0.59 μ Au (1) Z ¯ = Z Cu 2.94 + 2.26 Z Se 2.94 + 1.75 Z Au 2.94 2.94 (2)which both are higher than CuSe or Au NPs. The signal intensity demonstrated much more powerful consequences than commercial iopromide in CT imaging. The demonstrated synergetic effects from engineered high- and low-Z elements for enhanced imaging effect opens a path for the new design of materials. Bismuth-based NPs Compared with Au, bismuth (Bi) has particular advantages in price and is also a suitable candidate for CT imaging (Z = 83, absorption edge kBi = 90.5 KeV). As one of the most biocompatible elements,58 it has been increasingly applied in biology as a diagnostic agent (CT imaging) and therapeutic material (photothermal therapy and radiotherapy). The primary Bi-based NPs CT contrast agents were proposed by Rabin et al. in 2006.59 The produced polyvinyl pyrrolidone (PVP)-coated Bi2S3 with 10–50 nm in length and 4 nm in thickness significantly enhanced the visibility of vasculature and internal organs. However, the controllable synthesis of Bi-containing NPs still needs to be investigated compared with Au NPs, which impedes their development as a contrast agent. Our group60 explored large-scale oleic-acid-coated Bi2S3 nanodots with excellent monodispersity using bismuth neodecanoate as a precursor. The employment of oleic acid avoided the reduction of Bi3+ to metallic Bi, and together with neodecanoate in organic bismuth salt, ensured the uniform size (2∼3 nm) due to the strong binding to Bi3+. The as-prepared nanodots were further coated with PVP to improve hydrophilicity. The liver and spleen were still clearly delineated at 4 h after intravenous injection. No adverse effects were found, indicating low toxicity even at high doses. What is more, the large-scale synthesis constituted an essential step toward future clinical application. It is well known that controllable morphology is one of the great advantages of nanomaterials. Other shaped materials such as two-dimensional (2D) nanomaterials have attracted tremendous interest in various fields with the rapid development of grapheme.61 The reduced dimensionality makes for a conspicuous physical chemical property. Recently, the Shi’ group62 resolved the question about the crucial structure and compositional characteristics of 2D bismuthene in biological applications. An exquisite physical exfoliation strategy was introduced (Figure 3a). The sodium borohydride was used after a water-mediated freezing and thawing process for the designed 2D bismuthene nanosheets. The enhanced CT signals of well-engineered nanosheets increased over time after intravenous injection and clearly showed the position of the tumor. Meanwhile, the large surface area made good use of the optical performance to promote phototherapy effectiveness. Figure 3 | (a) Schematic illustration of 2D ultrathin bismuthene for CT imaging in vivo, and the horizontal section images of the treated mouse (red circle denotes tumor position). (b) The synthesis process of the stable, uniform Yb-based NPs and the CT imaging effect in vitro. Reprinted with permission from ref 62. Copyright 2020 WILEY-VCH; ref. 63. Copyright 2012 WILEY-VCH. Download figure Download PowerPoint Ytterbium-based NPs Ytterbium-based NPs hold great promise in CT imaging for the following reasons: (1) the appropriate K-edge (absorption edge kYb = 61 KeV) is exactly right in the higher-intensity region of the X-ray spectrum with a higher atomic number (ZYb = 70), which ensures the stronger contrast under lower radiation exposure; (2) the abundant contents in the Earth crust compared with Au and Bi makes it possible for industrial production; (3) the favorable safety and stability profiles have been demonstrated in some research, and the low toxicity and metabolizability promote the evolution in vivo.64,65 We reported the Yb-based NPs CT contrast agents for the first time.63 The controllable size and surface modification was realized by dispersing them in oleic acid, which inhibited the agglomeration. The surface was then modified with biocompatible polymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-{methoxy-[poly(ethylene glycol)]-2000} (DSPE-PEG2000) for further applications (Figure 3b). The NPs obtained showed excellent imaging capability owing to the proper kYb values under general operating conditions (120 kVp), which was nearly twice the intensity compared with iobitridol. The upconversion luminescence images were also investigated, and the internalized by Hela cells was concerned with concentration of nanoparticles. The prolonged circulation time and hypotoxicity satisfied the requirements for CT imaging. As the well-known characteristic of upconversion photoluminescence, Yb-based NPs can convert long wavenumber light in the near-infrared (NIR) region into visible light,66 leading to the combination of CT imaging and deep phototherapy. This endows the Yb-based NPs with a more effective treatment property, easily achieving the integration of diagnosis and therapy. Yuan’s group26 described an integrated platform including photosensitizer Ce6 and Yb-based upconversion core–shell nanostructures. Ce6 was activated by the light emitted by unconversion nanostructures under the irradiation of NIR laser and produced reactive oxygen species for deep phototherapy. In the meantime, the bright CT signal after injection revealed the tumor in sharp contrast to the surrounding tissue. Other metal-based NPs In recent years, the flourishing progress of NP research has made good use of various chemical elements with the aim of different recommendations. It has enriched the variety of materials used for contrast agents. Apart from the contrast agents mentioned earlier, there are still numerous elements which possess high atomic numbers and suitable absorption edges, being investigated for CT imaging. Moreover, biocompatibility, stability, and excellent contrast intensity are critical factors that need to be considered, for example, with regard to tantalum (ZTa = 73), silver (ZAg = 47), platinum (ZPt = 78). Simultaneously, it is obvious that these materials generally possess the unique ability to implement different functions, overcome the disadvantage of traditional contrast agents, and give full play to the superiority of CT imaging. Many researchers are focusing on the optimization design of these NPs.28–30,67 Here, we list such metal-based NPs and are optimistic about their enormous potential as contrast agents (Figure 4). Figure 4 | The recent development of different metal-based NPs for enhanced CT imaging, which can effectively attenuate X-ray and indicate the location of tumor clearly. Reprinted with permission from ref 67. Copyright 2017 WILEY-VCH; ref 28. Copyright 2018 The Royal Society of Chemistry; ref 29. Copyright 2016 WILEY-VCH; ref 30. Copyright 2019 WILEY-VCH. Download figure Download PowerPoint Perovskite NPs for X-ray Imaging The optimization of contrast agents for CT imaging should be considered not only for the ability to attenuate elements, but also for the improved sensitivity to X-ray. Perovskite, as an emerging material, has shown significant development in the field of photovoltaics.68 It is worth noting that the characteristics such as the high atomic number, large carrier mobility and lifetime, and ease of solution processing make it an ideal material for direct X-ray imaging.69–71 Figure 5a illustrates the process of X-ray imaging by a perovskite-based flat-panel system.72 The X-ray attenuated by hand can be captured by a detecting panel and creates an intensity distribution, which is further proportionally converted to an electronic signal, and finally read out by pixel detector. Though this material has not been widely used in CT imaging, the progress in high sensitivity and low detection limit for X-ray imaging make it promising in the future. Figure 5 | (a) The schematic diagram of the flat-panel X-ray imaging system. (b) The schematic illustration of the imaging process. (c) The optical and X-ray images of the logo acquired by the linear detector array. Reprinted with permission from ref 72. Copyright 2020 WILEY-VCH; ref 73. Copyright 2019 the Springer Nature. Download figure Download PowerPoint Tang’s group73 reported a Cs2AgBiBr6 nanocrystal that solved the toxicity problem of Pb-based perovskite and largely eliminated the ionic migration in polycrystalline wafers (Figures 5b and 5c). The as-prepared perovskite wafers exhibited low noise and high sensitivity to X-ray, demonstrating strong competitiveness as next-generation X-ray imaging flat panels. In other words, the increased sensitive response to X-rays means the use of a lower dose of X-ray, which is closely related to safety during CT imaging. Except for converting X-rays into electronic signals, Yang’s group74 designed a series of perovskite nanocrystals as flexible, high-sensitivity X-ray detectors. The perovskite comprising caesium and lead atoms exhibited strong X-ray absorption and intense radioluminescence at visible wavelengths. What is more, the visible spectrum was tunable by tailoring the anionic component during the synthesis. Although the existence of Pb is potentially toxic for the human body, the high sensitivity to X-ray and the induced emission of visible light open a new door to expand the application of CT imaging and combine with fluorescence (FL) imaging. The combination with other imaging modalities With the advance of modern medicine and its armamentarium, the increase of imaging methods has facilitated the diagnosis of disease. Though X-ray CT imaging is a robust, reliable, and most frequently used imaging method with high resolution and high efficiency, there are still undeniable disadvantages. For example, X-ray CT imaging is not applicable for the examination of hollow viscus such as the gastrointestinal tract. It is also powerless when the imaging of variations of metabolism and functions are required. Similarly, despite remarkable high spatial resolution and soft tissue contrast, MRI is limited by the hyposensitivity to bone damage.75 The recent evolution of imaging technology allows the hybrid scan to combine multiple imaging modalities.76 There is no doubt that the endogenous properties of conventional contrast agents are not supposed to function in multiple imaging modalities, resulting in unilateral information at the time of diagnosis, especially in the initial stages of the disease. On the other hand, the administration of various contrast agents increases the potential toxicity and side effects. Fortunately, the development of NPs has led to a diversity of nanomaterials in combination with various elements, such as Fe for MRI, Gd for CT, 64Cu for PET. With superior physical and chemical properties, they are expected to unleash the potential of multiple imaging modalities and compensate for the deficiency of each other. With MRI MRI provides an important noninvasive tool for diagnostic imaging, resulting from its distinct soft tissue contrast, high spatial resolution, and nonradiative process.77–79 The disadvantage of MRI, which lacks discrimination ability in bone, happens to be the strength of CT imaging. However, clinically used MRI contrast agents that are nonfunctional (iron-based Feridex, approved in 1996; manganese-based Teslascan, approved in 1997) or extremely unsatisfactory in CT imaging (gadolinium-based gadolinium-diethylenetriaminepentaacetic (Gd-DTPA), approved in 1988) are useless in MRI. Likewise, clinically used iodine-based CT contrast agents cannot be used in MRI. The multifunctional NPs allow for the combination of MRI and CT, which improves the resolution, sensitivity, and accuracy of the resulting diagnostic images. Lanthanide-based NPs have drawn lots of attention as multimodal imaging contrast agents due to their unique characteristics. Ln3+ such as Gd3+ and Dy3+ can be utilized to accelerate the nuclear relaxation for MRI80 and their high atomic numbers ranging from 57 to 71 also indicate their potential for CT imaging. What is more, similar chemical properties make it easier to integrate. Almutairi’s group81 reported heteroepitaxial NPs for dual-modal CT/MRI imaging. The engineered interfacial NaLuF4 layer thickness decelerated the tumbling of Gd3+ for enhanced MRI imaging. Lu3+ ensured the highly efficient X-ray attenuation with 70% higher contrast compared with clinical iodine-based contrast agents (Figure 6a). The NPs described confirmed the multimodal enhanced imaging resulting from the synergistic effect and demonstrated their enormous potential in diagnostics. Figure 6 | (a) The schematic diagram of enhanced imaging by lanthanide NPs (left) and the CT imaging (middle), MRI imaging (right) in vitro under different thicker interfacial. (b) The synthesis process of Gadolinium-based NPs contrast agents (top) and the MR imaging (bottom left) and CT imaging (bottom right). Yellow dashed circles show the difference in size, green arrows indicate the obvious signal at different times, and red arrow indicates indiscernibility. Reprinted with permission from ref 81. Copyright 2017 American Chemical Society; ref 82. Copyright 2019 WILEY-VCH. Download figure Download PowerPoint NPs based on one element with simultaneous CT and MR contrast-enhanced properties have been the subject of a great deal of research, which may lighten the burden during the design and synthesis process. Considering the high atomic number (ZGd=64) of Gd, it can examined for use in “one for all” NPs contrast agents.74 Walboomers and colleagues82 developed surface-functionalized Gd2O3 NPs (Figure 6b). The synthesized Gd2O3 (<5 nm in diameter) was encapsulated in 3-glycidyloxypropyl trimethoxysilane to stabilize it in aqueous solution and further functionalize it with bisphosphonates.82 Bisphosphonates were utilized to target bone and prolong the residence time for better imaging efficacy. The ultrasmall size and hydrophilia ensured the direct interaction and rapid exchange with the surrounding water, resulting in the brightness T1 contrast. And the enhanced CT signal intensity persisted in site even 8 weeks postsurgery. It is worth mentioning that Lu’s group83 also proposed gallium microparticles for enhanced antitumor cryoablation. They discovered the influence on T2-weighted MRI of gallium for the first time. Taking the excellent X-ray absorption into account, gallium-based NPs also possess great potential for the combination of CT and MRI in clinical use. With PET PET is an advanced technology in the field of nuclear medicine.84 It has shown potent ability in preclinical and clinical diagnosis with high sensitivity and excellent temporal resolution, PET’s most attractive advantage is the quantitative information about targeting efficiency and pharmacokinetics that it provides.85 In spite of this, PET is also limited by its low spatial resolution, which remains a severe hindrance in disease diagnosis.84 This problem can be well addressed by the synergistic imaging modalities. Townsend and Cherry86 combined CT and PET for the first time in 2001, which rapidly gained recognition as a speedy, whole-body, quantitative, and qualitative diagnostic tool in oncology. Therefore, the research of PET/CT bimodal contrast agents is highly requ