Advances on Nanomedicines for Diagnosis and Theranostics of Hepatic Fibrosis

Hepatic fibrosis is a pathological condition after chronic hepatic injury. As liver is an organ with high regenerative capacities, most acute hepatic injuries would cause acute damage to the hepatic parenchymal tissue, but complete recovery of the hepatic tissue and its original architecture can be achieved after acute injury elements have been eliminated. However, although hepatic fibrosis is usually reversible, fibrotic lesions at a late stage are irreversible. Without proper management to eliminate acute injury factors, the current fibrosis status of the liver would not be maintained but continues to be exacerbating. The progression usually leads to the formation of pseudolobule and irreversible changes in the hepatic tissue, i.e., hepatic cirrhosis. It has been well accepted that hepatic cirrhosis is precancerous lesions of hepatic carcinoma. Thus, hepatic fibrosis is not an isolated disease, but it is an intermediate disease that may lead to the formation of hepatic cirrhosis and eventually the dysfunction of this whole hepatic tissue and the formation of the hepatic carcinoma. Although there are no current data on the epidemiology of hepatic fibrosis, a recent study has indicated that hepatic cirrhosis ranks 13th in terms of the lost life years with estimated 1.2 million deaths annually, and hepatic carcinoma ranks 21st with 818 000 deaths. Therefore, early and accurate diagnosis and proper management of evolution of hepatic fibrosis due to chronic hepatic injury into hepatic cirrhosis/carcinoma are crucial to improve the life quality of these patients and reduce the casualties from hepatic cirrhosis/ carcinoma. However, early and accurate diagnosis of hepatic fibrosis is a rather challenging task that has not been achieved in the clinical practice. Current diagnostic methods of hepatic fibrosis often suffer from low sensitivity and specificity, interference from other complications, and poor efficacies when they are applied at an early stage of this disease, and design and development of new effective diagnostic methods and probes are in great demand. Recently, structures, sizes, and surface functional groups of nanomedicines have been manipulated and optimized for early and targeting diagnosis and effective treatment of many chronic or malignant diseases with reduced side effects. Promising application of nanomedicines for diagnosis and theranostics of hepatic fibrosis as a chronic hepatic disease has been X. Dai, Dr. Y. Zeng, Prof. H. Zhang, Prof. Q. Gong, Prof. K. Luo Huaxi MR Research Center (HMRRC) Department of Radiology Functional and molecular imaging Key Laboratory of Sichuan Province West China Hospital Sichuan University Chengdu 610041, China E-mail: tzeng92@foxmail.com; luokui@scu.edu.cn X. Dai West China School of Medicine Sichuan University Chengdu 610041, China Prof. H. Zhang Amgen Bioprocessing Centre Keck Graduate Institute CA 91711, USA Prof. Z. Gu, Prof. Q. Gong, Prof. K. Luo Research Unit of Psychoradiology Chinese Academy of Medical Sciences Chengdu 610041, China


Background of Hepatic Fibrosis
Hepatic fibrosis is a pathological condition after chronic hepatic injury. As liver is an organ with high regenerative capacities, most acute hepatic injuries would cause acute damage to the hepatic parenchymal tissue, but complete recovery of the hepatic tissue and its original architecture can be achieved after acute injury elements have been eliminated. However, although hepatic fibrosis is usually reversible, fibrotic lesions at a late stage are irreversible. [1] Without proper management to eliminate acute injury factors, the current fibrosis status of the liver would not be maintained but continues to be exacerbating. [2] The progression usually leads to the formation of pseudolobule and irreversible changes in the hepatic tissue, i.e., hepatic cirrhosis. [3] It has been well accepted that hepatic cirrhosis is precancerous lesions of hepatic carcinoma. [4] Thus, hepatic fibrosis is not an isolated disease, but it is an intermediate disease that may lead to the formation of hepatic cirrhosis and eventually the dysfunction of this whole hepatic tissue and the formation of the hepatic carcinoma. Although there are no current data on the epidemiology of hepatic fibrosis, a recent study has indicated that hepatic cirrhosis ranks 13th in terms of the lost life years with estimated 1.2 million deaths annually, and hepatic carcinoma ranks 21st with 818 000 deaths. [5] Therefore, early and accurate diagnosis and proper management of evolution of hepatic fibrosis due to chronic hepatic injury into hepatic cirrhosis/carcinoma are crucial to improve the life quality of these patients and reduce the casualties from hepatic cirrhosis/ carcinoma. However, early and accurate diagnosis of hepatic fibrosis is a rather challenging task that has not been achieved in the clinical practice. Current diagnostic methods of hepatic fibrosis often suffer from low sensitivity and specificity, interference from other complications, and poor efficacies when they are applied at an early stage of this disease, [6] and design and development of new effective diagnostic methods and probes are in great demand. Recently, structures, sizes, and surface functional groups of nanomedicines have been manipulated and optimized for early and targeting diagnosis and effective treatment of many chronic or malignant diseases with reduced side effects. [7] Promising application of nanomedicines for diagnosis and theranostics of hepatic fibrosis as a chronic hepatic disease has been also demonstrated, as shown in Table 1. These nanomedicines target specific biomarkers of hepatic fibrosis, such as hepatic stellate cells (HSCs) and collagen, to achieve early and accurate diagnosis of this disease. This review summarizes recent advances of nanomedicines in diagnosis and theranostics of hepatic fibrosis.

General Pathology of Hepatic Fibrosis
Hepatic fibrosis occurs when the liver is encountered with chronic, persistent, or repetitive injuries. These injuries are beyond the regenerative capacity of the organ to repair the damaged tissue, resulting in abnormal inflammatory infiltration with abnormal wound healing responses. The hepatic tissue is replenished with extracellular matrices (ECMs) generated around the damaged tissue, rather than fresh tissues, after stimulation by acute injuries. The accumulation of ECMs leads to the formation of fibrous scars. These fibrous structures can disrupt the normal architecture of the liver, ultimately resulting in the dysfunction of the whole organ. [8] ECMs mainly include collagen type I and III. This process has been demonstrated to be mediated by activated HSCs (aHSCs) which are transdifferentiated from quiescent HSCs (qHSCs) under stimulation of continuous chronic hepatic injuries ( Figure 1). [9] Tsuchida et al. have suggested several signal pathways that are involved in the activation of HSCs, including transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF), [10] and these pathways induce HSCs to present the fibrogenic effect. Among them, one of the key signal pathways is the TGF-β-based pathway. It has been reported that TGF-β could directly activate the Janus kinase 1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) pathway through its binding to JAK1. This binding would initiate a primary SMAD-independent process and another secondary SMAD-dependent process to phosphorylate STAT, leading to the activation of HSCs. [11] TGF-β has also been found to interact with TGF-β-activated kinase 1 (TAK1), [12] induce the expression of Forkhead box O 3a (FoxO3a), [13] and regulate the plasticity of hepatic macrophages. [14] All these effects of TGF-β would lead to HSC activation and collagen production. Apart from hepatic fibrosis, TGF-β is involved in nephrotic fibrosis as well. [15] Other pathways including the Hedgehog (Hh) ligand and its receptor smoothened homolog (SMO) may also be involved with the activation of HSCs. Both G protein-coupled receptors of HSCs and epigenetic signals (microRNAs, DNA methylation, and histone modification) could activate and inactivate HSCs. Innate immune signaling would also take part in either activation or inactivation of HSCs depending on Toll-like receptors (TLRs) and different cytokines involved. In addition, it has been reported that oxidative stress is another important element that would result in the activation of HSCs. Several receptors such as integrin α v β 3 are expressed on the surface of aHSCs, which could be potential targeting biomarkers of aHSCs. [16] Studies have designed diagnostic and therapeutic agents to especially target integrin α v β 3 for detection and treatment of hepatic fibrosis, [17] but the efficacy of these novel agents is still not high enough to be accepted in the clinical practice. During a pathological course of hepatic fibrosis, these aHSCs would secrete ECMs into different locations of the hepatic parenchymal tissue and the Theranostic a) (c)RGD, (cyclic) arginine-glycine-aspartic acid; HSC, hepatic stellate cell; USPIO, ultrasmall superparamagnetic iron oxide; ICG, indocyanine green; PLGA, poly(lacticcoglycolic acid); PFOB, perfluorooctyl bromide; SPION, superparamagnetic iron oxide nanoparticle; EPR, enhanced permeability and retention; PONI, poly(oxanorborneneimide); ELF test, Enhanced Liver Fibrosis test; VA, vitamin A; T-PBP, polyethylene glycol-polyethyleneimine-poly(N-(N 0 ,N 0 -diisopropylaminoethyl)co-benzylamino) aspartamide; RLX, relaxin; HBc, hepatitis B core; QGd, quercetin-gadolinium complex. location depends on the types of chronic injuries that induce cell activation. Chronic hepatitis, chronic cholestasis, and hemochromatosis result in the deposition of ECMs around the portal vein and its branches to form the portal-based hepatic fibrosis. By contrast, alcoholic or nonalcoholic fatty liver diseases and venous outflow obstruction would trigger the deposition of ECMs around the central veins, leading to the central-based hepatic fibrosis. [18] Kupffer cells, another major type of cells for the formation of hepatic fibrosis, are hepatic macrophages that mediate regenerative responses due to acute hepatic injuries. During the chronic hepatic injury process, Kupffer cells activate HSCs to cause fibrogenesis and release CCL-2 and CCL-5 to stimulate the infiltration of bone marrow-derived immune cells. [19] Most of these immune cells are monocyte-derived Ly6C hi macrophages that could promote the fibrogenesis process. [19,20] It is noted that these Ly6C hi macrophages are not always harmful. Protective Ly6C hi macrophages transdifferentiated from HSCs may reverse the fibrogenic microenvironment into a fibrosis  [109,110] Copyright 2011, Elsevier Ireland Ltd. and Copyright 2018, Elsevier Ireland Ltd., respectively.
www.advancedsciencenews.com www.advnanobiomedres.com resolution-favored environment to promote the resolution of the generated ECMs (e.g., collagen). [2] It has been reported that the fractalkine receptor CX 3 CR-1 is probably engaged in the transdifferentiation of profibrogenic macrophages into proresolution macrophages, [21] which could provide a new therapeutic approach to treating hepatic fibrosis. Myofibroblasts which originally reside in the portal tracts could also transform cholangiocytes into ECM-producing cells for fibrogenesis in the portal areas. [22] With the development of the fibrogenic process, these ECMs further evolve into fibrous septa. The septa eventually leave the vascular area where they are originally formed and migrate to other vascular areas nearby, leading to severe sinusoidal/perisinusoidal changes such as obstructing the substance exchange between sinusoids and hepatocytes. [23] 3

. Current Diagnosis Methods for Hepatic Fibrosis
Although it is critical to identify and prevent primary hepatic changes caused by chronic hepatic injuries, due to the fact that hepatic fibrosis could be induced by a variety of pathogenic elements and hepatic injuries, it is more important to identify early fibrous changes in the hepatic tissue and conduct effective management to prevent fibrosis from progression into cirrhosis or carcinoma that are irreversible. Currently, the gold standard for diagnosing and staging hepatic fibrosis is to conduct liver biopsy. When the extracted hepatic parenchymal is examined under a microscope, one could directly see formed collagens and structure changes of the hepatic tissue to diagnose and determine the stages of hepatic fibrosis. The development of hepatic fibrosis histopathology has enabled establishment of a scoring system that could help standardizing histological definition and stages of hepatic fibrosis. This widely used scoring system can be used to evaluate both the inflammatory characteristics and fibrosis-resulted hepatic architectural disruption. [24] It was first introduced in 1981 that the grade and architectural disruption by fibrous elements are assessed through the evaluation of periportal necrosis, intralobular necrosis, and portal inflammation to determine the stage of hepatic fibrosis. [25] However, liver biopsy is an invasive procedure that could cause pain to patients and may not be able to be conducted if certain complications or coexisting diseases are present in patients. [26] In addition, this invasive procedure may also generate procedure-related complications, such as bleeding at the biopsy site, inflammation dissemination, or even possible tumor seeding if patients have already had small hepatic tumor masses without notice. [27] In addition, as fibrosis in the liver cannot spread into a wide region at an early stage and liver biopsy samples only account for nearly 1/50 000 of the whole liver tissue, the fibrosis-generated site could be missed, leading to false-negative diagnosing results for hepatic fibrosis. [27] Serum markers can be utilized to help the diagnosis of hepatic fibrosis. [28] It has been reported that hyaluronic acid, [29] ferritin, [30] Mac-2 binding protein glycan isomer, [31] interleukin-8, osteopontin, and monocyte chemoattractant protein-1 [32] along with other serum markers have been demonstrated to help evaluating the fibrosis of liver. Clinically and commercially, at least four serum markers systems have been widely applied, including FibroTest/FibroSure, [33] Hepascore, [34] FibroSpect, [35] and Enhanced Liver Fibrosis (ELF). [36] These serum markers could be acquired through noninvasive procedures; thus, the diagnosis method based on serum markers is more acceptable by patients. More serum markers are emerging but they are still under study. [37] One critical issue of these methods is that most of these serum markers are not exclusive for hepatic fibrosis and some of them can only indirectly indicate the dysfunction of the liver. Therefore, it is very challenging to accurately assess the degree of hepatic fibrosis by the presence or any changes in these serum markers because they may be confounding factors from other hepatic or extrahepatic diseases. [38] Another issue of this diagnosis method is that most of these serum markers are reported to be diagnostically effective only in the presence of hepatitis C or B infection or a fatty liver, [39] which means these serum markers may not be able to detect fibrotic tissues that are not caused by these diseases. Thus, reliable and reproducible serum markers need to be explored to predict and monitor hepatic fibrosis.
Another noninvasive method for assessing hepatic fibrosis is through medical imaging. Ultrasound elastograph (USE) and magnetic resonance elastograph (MRE) are two most widely used imaging methods for diagnosing hepatic fibrosis. Both methods apply a radiation pressure onto the hepatic tissue and assess the response of the liver to this external pressure. It has been reported that MRE has a high resolution and a high sensitivity, and it also has a standardized scan protocol and it is independent of technicians; [40] therefore, MRE has a better performance in terms of the accuracy of diagnosing hepatic fibrosis compared with USE. Unfortunately, a high cost and the complexity of scanning sequences of MRE restrict its use in comparison with USE. [41] Other imaging methods are helpful in diagnosing and assessing hepatic fibrosis, but they have less applications than elastographs, including diffusion weighted imaging (DWI), [42] magnetic resonance imaging (MRI) T 1 and T 2 mapping, [43] MRI perfusion (PWI), [44] texture analysis (TA), [45] and susceptibility weighted imaging (SWI). [46] Indeed, some studies have suggested that these imaging methods, especially MRE, could reach an equivalent or even better efficacy than liver biopsy and they could be used to replace the invasive diagnostic method. However, more imaging studies on hepatic fibrosis have revealed that these current clinical practical imaging methods have not achieved great performance in the early diagnosis of hepatic fibrosis, [6,47] whereas the early stage of hepatic fibrosis is a crucial time to obtain satisfactory therapeutic effects for reverse hepatic damages. [48] Overall, liver biopsy, serum biomarkers, and imaging methods have been applied for diagnosis and staging of hepatic fibrosis. But each of these diagnostic methods has their drawbacks. Low sensitivity and specificity, interference from other complications, and poor efficacies of these methods have been the driving forces for exploring new methods or improving current methods for diagnosis of hepatic fibrosis, especially for that at an early stage. Recently, nanomaterials have been developed for the diagnosis of hepatic fibrosis due to their easiness of production, preferable biocompatibility, and most importantly, flexibility in incorporating targeting groups. [49] By carefully designing a backbone structure and a hepatic fibrosis-targeting moiety, these novel nanomedicines could specifically detect pathological elements of hepatic fibrosis, such as HSCs (Section 4), pathological ECMs (Section 5), and Kupffer cells and other fibrogenesis-related receptors on hepatocytes (Section 6). As these pathological elements are present at an early stage of the course of hepatic fibrosis, these targeting diagnostic probes could not only detect hepatic fibrosis in an enhanced specificity, but also achieve the detection at an early time. Nanomedicines could also incorporate therapeutic agents toward fibrotic tissues to achieve the theranostics of hepatic fibrosis (Section 7). This could increase the reverse rate of fibrotic lesions and also prevent hepatic cirrhosis and carcinoma.

Nanomedicines Targeting HSCs
As HSCs are one of the major cells that play a crucial role in the formation and progression of hepatic fibrosis, nanotechnology has been applied to help targeted imaging of HSCs. Although there are many biomarkers expressed by HSCs, integrin α v β 3 is a primary target for nanomedicines because this integrin activates TGF-β, a key profibrogenic cytokine, to induce hepatic fibrosis. [50] Zhang et al. [51] used an arginine-glycine-aspartic acid (RGD) peptide to modify ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to form a HSC-targeting T 2 contrast agent. They applied this contrast agent in a CCl 4 -induced hepatic fibrosis animal model for MRI scans and they have found that RGD-USPIO could achieve molecular imaging of α v β 3 expressed on activated HSCs. This method could have great potential for defining different stages of hepatic fibrosis. Li et al. [52] integrated Fe 3 O 4 nanoparticles, indocyanine green (ICG), and RGD to construct SPIO@SiO 2 -ICG-RGD dual-modality nanoparticles (Figure 2A). After they were applied for MRI and near-infrared (NIR) fluorescence imaging both in vitro and in vivo on CCl 4induced hepatic fibrosis mice, these core/shell nanoparticles could specially target integrin α v β 3 expressed on HSCs with unique MRI and NIR signals for hepatic fibrosis. Their study also suggested that these SPIO@SiO 2 -ICG-RGD dual-modality nanoparticles were effective in identifying hepatic fibrosis at an early stage. [52] In addition to T 2 imaging probes for hepatic fibrosis, a dendrimer nanoprobe has been introduced to achieve T 1 imaging of fibrotic tissues ( Figure 2B). [53] In this nanoprobe, the cyclic RGD peptide was used as the targeting group toward activated HSCs, and strong T 1 contrast signals were observed after the gadolinium (Gd)-incorporated dendrimer was distributed in the fibrotic tissues. The signal intensity was in a positive correlation with the development and progression of hepatic fibrosis, demonstrating that this noninvasive method could be used to define the fibrosis staging. Reproduced with permission. [52] Copyright 2018, The Royal Society of Chemistry. Reproduced with permission. [53] Copyright 2016, Elsevier. Reproduced under the terms and conditions of the Creative Commons Attribution 4.0 International License. [111] Copyright 2015, The Authors, published by Springer Nature.
www.advancedsciencenews.com www.advnanobiomedres.com Based on the similar targeting mechanism, a core-shell nanostructure that combined the cyclic RGD peptide and an ultrasound agent was introduce. [54] In this structure, perfluorooctyl bromide (PFOB) acted as a core and cyclic RGD-modified poly(lacticco-glycolic acid) (PLGA) as a shell. This nanoprobe was able to display great contrast echo intensity for the ultrasound images to distinguish fibrotic tissues from normal hepatic tissues. Owing to affinity of the cyclic RGD peptide to integrin α v β 3 , this nanoprobe could effectively target HSCs in a dosedependent manner and the dose increased in accordance with the development course of the hepatic fibrosis disease, suggesting this method may be useful in characterizing this disease at different stages. More recently, this aHSCs-targeting cyclic RGD peptide was also introduced to therapeutic liposomes that could result in the alleviation of hepatic fibrosis. [55] As previous studies [56] have demonstrated the potential of liposomes to conjugate the Gd complex or iron oxide nanoparticles to construct nano-based contrast agents for MRI, cyclic RGD-guided liposomes could be explored as a T 1 /T 2 dual hepatic fibrosis imaging probe with the ability of theranostics for hepatic fibrosis. Although [ 64 Cu]CuS nanoparticles, [57] 99m Tc labeled NAD/monosaccharide-coated ferrihydrite nanoparticles, [58] rare-earth-doped nanoparticles, [59] or paramagnetic liposomes [60] have been developed for imaging of integrin α v β 3 , their application is not for hepatic fibrosis. They could be evaluated for identifying and characterizing hepatic fibrosis.
The specificity of α v β 3 -targeted nanomaterials could be the major issue for the detection of hepatic fibrosis. As other diseases such as hepatic carcinoma could cause an increase in integrin α v β 3 , the diagnosis results for the imaged lesions from these nanomaterials may not be definitely applied to hepatic fibrosis. Fortunately, hepatic fibrosis and hepatic carcinoma are at different stages of this disease, and these nanoparticles could be applied for diagnosing hepatic fibrosis at the early stage before it progresses into hepatic cirrhosis or carcinoma. The metabolism of USPIO nanoparticles in the animal model was also found to have a confounding impact for the diagnosis of this disease according to the study by Zhang et al. Modification of these USPIO nanoparticles to avoid this metabolic issue but maintenance of the targeting effect toward integrin α v β 3 need to be addressed in the future studies. [51] Apart from integrin α v β 3 , another biomarker option is cluster determinant 44 (CD44). CD44 is a hyaluronic acid receptor expressed on the surface of HSCs. As hyaluronic acid, one of the fibrogenesis biomarkers, significantly increases in the fibrotic tissues, [61] the expression of CD44 would increase accordingly during the course of the disease. [62] Hyaluronic acid-conjugated nanomaterials have been applied as livertargeting imaging probes and they may have the potential for imaging of hepatic fibrosis. [63] Hyaluronic acid-based nanomedicines could also achieve targeted delivery of therapeutic agents for the treatment of hepatic fibrosis, [62b] and they could be utilized as theranostic agents for the management of this chronic hepatic disease.
Other biomarkers of HSCs, such as translocator protein, desmin, and vimentin, have been reported to have the potential to be the target for HSC imaging. Contrast agents, 18 F-FEDAC [64] and 99m Tc-GlcNAc-PEI, [65] may be applicable for imaging HSCs. Compared with current diagnostic methods for hepatic fibrosis, the use of nanomedicines with targeting groups could specifically recognize HSCs. As HSCs are the key drivers for initiating the fibrogenic process and their activity is positively correlated with the degree/stage of hepatic fibrosis, these HSC-targeting nanomedicines could not only help defining the staging of hepatic fibrosis, but also have the potential to identify fibrotic lesions before a large amount of ECMs are accumulated. [66] However, due to a limited number of studies on these biomarkers, more investigations are in need to develop these biomarker-based nanomaterials for imaging HSCs.

Nanomedicines Targeting Extracellular Matrix
Another important target for imaging of hepatic fibrosis is to focus on the product of fibrogenesis, ECMs. [67] From the diagnostic imaging point of view, collagen is the most studied target among ECMs. Caravan et al. [68] first introduced a cyclic peptide, EP-3533, to specifically target type I collagen. They combined this low-molecular-weight cyclic peptide with the Gd complex to construct a novel Gd-based MRI contrast agent. This contrast agent have been demonstrate with great performance for imaging of many fibrogenic diseases [69] including hepatic fibrosis. [70] More studies [71,72] have revealed that this EP-3533-Gd-based contrast agent could not only detect the presence of hepatic fibrosis, but also have the ability to determine the stage of the hepatic fibrosis progression. One should note that despite its effectiveness in MRI, one study [71] has reported the side effect of EP-3533 because it retained in bone and other tissues. Apart from EP-3533, gold (Au) nanoparticles have also been explored for targeted imaging of collagen. Zhou et al. [73] constructed CNA35labeled perfluoropentane nanoparticles. These nanoparticles could specifically target fibroblasts and collagen. These nanoparticles were transformed from liquid to gaseous microbubbles after interaction with collagen, increasing the ultrasound contrast for ultrasound imaging of fibrotic tissues. Zhu et al. [74] synthesized anticollagen-I antibody-conjugated Au nanoparticles. These Au nanoparticles were applied to in vitro and in vivo CT scans, and collagen type I-targeting CT imaging of fibrotic tissues with high quality was demonstrated. Although two applications of Au nanoparticles were not for targeted imaging of hepatic fibrosis, these nanoparticles may be served as contrast agents for targeted imaging of hepatic fibrosis because the formation and progression of hepatic fibrosis are mainly involved with the engagement of fibroblasts and collagen. More studies are required by directly applying Au nanoparticles using hepatic fibrosis animal models.
Allysine exists in the ECMs in hepatic fibrosis and it could be served as another target for imaging methods. It has been reported [75] that allysine could stabilize the ECMs by conducting cross-linking condensation and it is one of the major components for fibrogenesis. To target allysine for imaging of fibrosis, Chen et al. [76] designed a hydrazide moiety-conjugated Gd-DOTA chelate (Gd-Hyd) contrast agent and demonstrated that this agent could specifically and effectively detect hepatic fibrosis and also determine the stages of its progression. Waghorn et al. [77] reported that allysine-binding Gd chelates (GdOA) displayed a higher relaxivity and a greater affinity to allysine compared with Gd-Hyd. They demonstrated that GdOA could identify and quantify the degree of fibrogenesis to help both initial diagnosis and the staging of the fibrotic disease. Wahsner et al. [ Ga-NODAGA-indole as an allysine-targeting probe for positron emission tomography (PET) of fibrogenesis. They have found that the accumulated amount of this PET probe in fibrotic tissues was 7 times higher than that of the control groups. They also discovered that the accumulation amount of this PET probe was in correlation with the concentration of allysine in the fibrotic tissues, which would allow to define the staging of this disease. Other studies have suggested targeted imaging of collagen remodeling by collagen hybridizing peptides [78] or elastin by Gd-ESMA, [79] but further studies are needed to confirm their efficacies for diagnostic imaging of hepatic fibrosis.
Compared with other hepatic fibrosis diagnosing methods, ECM-targeting probes could determine the stage of fibrotic lesions in a more direct manner. Current noninvasive diagnosis methods including elastography and serum sensors could help the detection of fibrotic lesions by measuring elastic properties and/or stiffness of the liver or identifying fibrosis-related serum factors; [80] however, the correlation between these properties or factors and fibrotic lesions is not very high and also these properties/factors may vary for each individual patient. [81] Whereas the ECM deposition is a basic pathological change for fibrosis, the detection of ECM accumulation in the liver by targeting nanomedicines could be a reliable diagnostic strategy by directly identifying the formation or determining the severity of hepatic fibrosis. However, ECMs include a complex matrix pool with many molecules. Currently, the role of each ECM molecule in the formation of hepatic fibrosis remains to be uncovered [82] and more potential ECM molecules could be explored to design specific nanomedicines for diagnosis of hepatic fibrosis.

Nanomedicines for Other Targeting Options
Although HSCs and ECMs are the main imaging targets for diagnosis of hepatic fibrosis, other targeting options have been explored to help imaging of fibrotic tissues. Saraswathy et al. prepared superparamagnetic iron oxide nanoparticles (SPIONs) stabilized by dextran and applied them to image hepatic fibrosis. [83] Although no specific targeting agents were conjugated in SPIONs, SPIONs could potentially target Kupffer cells in fibrotic tissues to display hypointense contrast in the lesions because a large number of Kupffer cells are present in fibrotic tissues in the liver compared with normal hepatic tissues [84] and most of SPIONs are metabolized through phagocytosis of Kupffer cells. [85] In addition, they suggested that these dextran-stabilized SPIONs could exhibit an enhanced permeability and retention (EPR) effect toward fibrotic hepatic tissues, thus enhancing their specificity and signal contrast at the lesions.
Another option to identify fibrotic lesions is to examine any change in receptors in the hepatic tissues. It has been previously reported that during the course of developing chronic hepatic diseases including fibrosis, several receptors would be up-or downregulated. For example, the number of the asialoglycoprotein receptor (ASGPR), highly expressed in healthy hepatocytes to remove galactose (Gal)-or N-acetylgalactosamine (GalNAc)terminated glycoproteins during their circulation to remain normal metabolism, [86] would be significantly decreased in the course of hepatic fibrosis. [87] In this context, several nuclear medicine contrast agents, such as a fluorine-18-labeled galactose derivative [ 18 F]FPGal, [88] 99m Tc-p(VLA-co-VNI), [89] 18 F-FBHGal, [90] and 111 In-hexavalent lactoside, [91] have been introduced as effective imaging probes for the detection of hepatic fibrosis lesions and staging of the disease course. But radiotoxicity of these contrast agents and a high cost of nuclear medicine examinations have significantly hampered their wide application clinically. To address these challenges, GalNAc-decorated nanostructured lipid carriers and PLGA-di-GAL nanoparticles were reported to achieve targeting internalization into hepatocytes through ASGPR recognition. [92] However, none of these ASGPR targeting nanomaterials have been examined for imaging of hepatic fibrosis, and evaluation studies of these nanomaterials for hepatic fibrosis will be encouraged from previous results for hepatocyte internalization.
In addition to nanomedicines for targeting these Kupffer cells and fibrogenesis-related receptors, recently, a NIR-II imaging probe based on a self-assembled small-molecule dye has been designed to visualize hepatic fibrosis lesions (Figure 3). [93] PEGylation was applied to help forming a nanosized probe from single molecules. This nanoprobe could be switched ON and OFF for the detection and assessment of hepatic fibrosis, and it was also applicable for imaging hepatic carcinoma and tracing the lymphatic system in the brain. However, the detailed mechanism for imaging of hepatic fibrosis by this nanoprobe was still unclear. It was assumed that the proliferation of connective tissues in the fibrotic liver would reduce the speed of the portal vein blood flow, resulting in the OFF signal in those fibrotic areas, [93] but more confirmation studies are still in need. In another study, a poly(oxanorborneneimide) (PONI) random copolymer scaffold sensory array was introduced for the early diagnosis of hepatic fibrosis. [94] Instead of a contrast agent for imaging of hepatic fibrosis, this array was developed as a serum biomarker sensor platform to achieve the ELF test. It was multiplexed with four fluorescent channels representing four ELF test biomarkers simultaneously from one single serum sample at a small volume. Furthermore, previous studies suggested that bioconjugates for detecting ELF biomarkers are unstable and often centralized pathology laboratories are required to conduct these tests, [95] but this PONI-copolymer sensing platform was robust, manageable, and rapid for generating ELF scores with sensitivity and specificity, both above clinical relevance thresholds. [94] However, large cohort studies are still needed to assess the clinical feasibility of this multichannel sensor array.
Although the nanomedicines for other targeting options could provide us different perspectives in designing novel diagnostic probes for hepatic fibrosis, these nanoprobes usually are very costly, and more studies are required to reveal their diagnostic mechanisms and confirm their efficacies in preclinical or clinical trials.

Theranostic Nanomedicines for Hepatic Fibrosis
The success of previous imaging of hepatic fibrosis also has inspired exploration of theranostic nanomedicines for this disease. By incorporating both imaging and therapeutic groups into one nanomedicine, it could effectively achieve both visualization and treatment of fibrotic lesions, contributing to www.advancedsciencenews.com www.advnanobiomedres.com monitoring treatment outcomes and reducing the incidence of side effects due to multiple injections of external nanomedicines. Kim et al. designed and synthesized a PEI/siRNA theranostic nanocomplex. [96] By conjugating N-acetylglucosamine, this nanomedicine could specifically target at desmin which was abundantly expressed on the surface of activated HSCs and be internalized by these fibrosis morbific cells. The internalized siRNA (transforming growth factor β1 siRNA, TGFβ1siRNA) was able to significantly reduce the expression of TGFβ1 and α-smooth muscle actin (α-SMA), the former of which is a fibrogenic cytokine that could lead to the activation of HSCs [97] and the latter of which is one of the main components in the fibrotic tissues. [98] Furthermore, this nanocomplex was also incorporated with ICG to achieve theranostics, which enabled fluorescence imaging of hepatic fibrosis tissues and monitoring of the treatment course. Similarly, Wu et al. prepared SPIO-decorated pHsensitive and vitamin A (VA)-conjugated copolymer micelles from VA-polyethylene glycol-polyethyleneimine-poly(N-(N 0 ,N 0diisopropylaminoethyl)-co-benzylamino) aspartamide (T-PBP). The micelles were used to deliver therapeutic microRNA-29b (miRNA-29b) and microRNA-122 (miRNA-122) against fibrosis to achieve synergic theranostics of hepatic fibrosis ( Figure 4A). [99] VA was used to enhance targeting HSCs and endocytosis of this nanomedicine, [100] and miRNA-29b and miRNA-122 were confirmed to reduce collagen production of HSCs [101] and SPIO enabled T 2 MR imaging. This novel nanomedicine demonstrated significant inhibition of fibrosisrelated gene expression and continuous monitoring through MRI scans, providing a safe and effective way for antifibrotic theranostics.
To increase the targeting ability and biosafety of relaxin (RLX) to treat the fibrotic liver, [102] Nagórniewicz et al. conjugated this antifibrotic drug onto the surface of dextran-coated SPION ( Figure 4B). [103] Dextran-coated SPION would not only significantly reduce the off-target effect of RLX, but also increase the circulation stability, improve pharmacokinetics toward hepatic absorption, and achieve multivalent interaction of RLX compared with monovalent interaction of free RLX, [104] which resulted in a better antifibrotic efficacy with reduced incidences of adverse effects. Furthermore, this RLX-SPION nanomedicine enabled T 2 MRI diagnosis of fibrotic tissues and monitoring of the entire therapeutic process, demonstrating a great potential for theranostics of hepatic fibrosis. More recently, hepatitis B core protein nanocages have been introduced to encapsulate the quercetingadolinium complex and/or labeled with NIR fluorescent probes ( Figure 4C). [105] These nanoparticles were applied to diagnostic imaging of hepatic fibrosis in the animal models through NIR fluorescent probes or MRI scans. It was found that by binding specifically to integrin α v β 3 , these nanoparticles could achieve targeted imaging of activated HSCs as well as suppression of proliferation and activation of activated HSCs. This dual effect suggested that these nanoparticles could serve as a contrast agent and a therapeutic agent for the antifibrotic theranostic strategy. A) Synthesis and self-assembly of a NIR-II imaging probe via PEGylation and B-E) its medical applications. This nanoprobe could not only achieve targeted imaging of fibrotic liver in an ON-OFF manner (B), but also have great potential for imaging of lymphatic vessels (C), cerebral blood vessels (D), and imaging-guided tumor surgery (E). Reproduced with permission. [93] Copyright 2018, Wiley-VCH.

Summary and Future Perspectives
Overall, there are many challenges in accurate diagnosis of hepatic fibrosis and the stage of the entire disease development course from chronic hepatic injury to hepatic fibrosis and hepatic cirrhosis/carcinoma, as well as targeted delivery of diagnosis agents into fibrotic tissues. Nanomedicines targeting HSCs, ECMs, fibrogenesis-related receptors, or serum biomarkers could significantly increase their diagnosis efficacy toward fibrotic tissues with high sensitivity and specificity. The targeting ability of these nanomedicines enables reduction in incidences of off-target events, promoting a safe way of detection of fibrosis.
Some of these nanomedicines have demonstrated their EPR effect toward fibrotic tissues and improve accurate diagnosis of hepatic fibrosis. The use of nanomedicines also allows the promotion of theranostics of hepatic fibrosis. Despite great achievements of these nanomedicines in various animal studies, a few challenging issues associated with nanomedicines still hamper translation of them into clinical trials. First, the intrinsic toxicity issue of nanomedicines is still haunting. Especially, many nanomedicines would be metabolized by the liver, leading to hepatotoxicity which could exacerbate the damage to hepatic tissues. [106] Thus, methods to reduce the toxicities and side effect incidences are still actively explored for allowing these diagnostic nanomedicines to be applied in clinical trials. Second, most of these diagnostic nanomedicines have been conducted on small animals, mostly on mice, but very few of them have been tested on large animal models, such as monkeys or pigs. In this context, the efficacies of these nanomedicines on small animals need to be confirmed in large animal models before they could be translated into human trials. [107] Third, clinical trial designs of these nanomedicines on hepatic fibrosis are also a challenge. One of unique benefits of using nanomedicines is to detect fibrotic lesions at an early stage. Unfortunately, early hepatic fibrosis are not easily diagnosable in patients in the clinical practice, and enrollment of these patients for clinical trials of nanomedicines could face a great challenge. [108] It is expected that development and modifications of nanomedicines and their efficacious data in large animal models will facilitate clinical transition of these nanomedicines into diagnostic products for the patients of hepatic fibrosis.  [103] Copyright 2019, The Authors, published by Elsevier Inc. (some rights reserved). C,D) Reproduced with permission. [105] Copyright 2019, American Chemical Society.