Liver Fibrosis: Underlying Mechanisms and Innovative Therapeutic Approach : A Review Article

Liver fibrosis is considered “a pathological repairing process in liver injuries leading to extracellular cell matrix (ECM) accumulation evidencing chronic liver diseases”. Chronic viral hepatitis, alcohol consumption, autoimmune diseases as well as non-alcoholic steatohepatitis are from the main causes of liver fibrosis (Lee et al., 2015; Mieli-Vergani et al., 2018). Hepatic stellate cells (HSCs) exist in the sinus space next to the hepatic epithelial cells as well as endothelial cells (Yin et al., 2013). Normally, HSCs are quiescent and mainly participate in fat storage and in the metabolism of vitamin A. HSCs are produced during liver injury and then transformed into myofibroblasts. The activated HSCs resulted in a sequence of events considered as marks fibrosis. The activation of HSCs mostly express alpha smooth muscle actin (a-SMA). Moreover, ECM is synthesized and secreted by HSCs that affects markedly the structure and function of the liver tissue leading to fibrosis (Tsuchida et al., 2017; Han et al., 2020). Hence, activated HSCs are attracting attention as potential targets in liver fibrosis. Many signaling molecules are involved in HSCs activation first and foremost, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-ß) (Tsuchida et al., 2017; Wang et al., 2020c) as interfering the PDGF or TGF-ß signaling pathways is a growing field for liver fibrosis treatment.

The ECM proteins' -particularly collagen type 1, -accumulation distorts the hepatic architecture through the formation of a fibrous wound, followed by the expansion of spherical areas of cells called nodules then hardening of the tissue. A series of events follows resulting in cirrhosis. Liver cirrhosis causes hepatocellular dysfunction, hepatocellular carcinoma (HCC) and hence, hepatic failure (Giannitrapani et al., 2014).
Liver fibrosis involves parenchymal, nonparenchymal liver cells and infiltrating immune cells. Cell death activates the inflammatory and pro-fibrogenic pathways that triggers fibrosis progression, as well as the possibility of reversing the process that aids in fibrosis resolution (Lee et al., 2015).
H e p a t i c m a c r o p h a g e s , i n j u r e d hepatocytes, lymphocytes and endothelial cells leads to the induction of HSC. The dead hepatocytes release of reactive oxygen species, damageassociated molecular patterns (DAMPs) which stimulate macrophages (Kupffer cells) to release proinflammatory biomarkers and pro-fibrogenic markrs, (Yang et al., 2012).
Other pro-inflammatory factors include gut-derived pathogen-associated molecular patterns (PAMPs) as well as chemokines like CCchemokine ligand-2 CCL2. CCL2, also known as monocyte chemotactic peptide-1 (MCP-1), it is considered a member of the beta (C-C) chemokine family. It is expressed in many cell types, including hepatocytes, stellate cells and inflammatory cells (Degre et al., 2012). C-C chemokine receptor type 2 (CCR2) has been found to be the known receptor for CCL2 and it is usually expressed on monocytes, T lymphocytes and basophils .
From the factors of chronic wound healing is the toll-like receptor 4 (TLR4) activation enhancing TGF-β-dependent HSC activation (Liu et al., 2014). Moreover, oxidative stress plays a very important role in all fibrogenic changes as shown by the overexpression of critical genes related to inflammation and chronic damage of tissues (de Castro Bras et al., 2020).
The increased activity of free radicals, together with the decreased antioxidant defenses result in the occurrence of oxidative stress that significantly contributes to fibrogenesis and excessive tissue remodeling (de Alwis et al., 2008).

Prevalence Of Liver Fibrosis In Egypt
Liver diseases with many types lead to liver fibrosis, which leads to further sedimentation of extracellular matrix followed by more complicated liver conditions that leads to death. According to the WHO data in 2018, Liver Disease Deaths in Egypt reached 12.40% of total deaths that ranks Egypt #1 worldwide (WHO, 2018). This report draws the scientists' attention to explore more new solutions to alleviate liver fibrosis.

Nanomedicine
Nanomedicine is concerned with nanoparticles'(NPs) designing and application in order to diagnose and treat diseases (Silva et al., 2019;Pucek et al., 2020). Nanomedicine is a critical field of nanotechnology research that has greatly affected and aided biomedicine through the past years. These nano formulations have been designed and adjusted as effective therapeutic agents to alleviate liver fibrosis targeting specific sites (Poilil Surendran et al., 2017). Moreover, nanostructures were designed as nano agents for contrast enhancement as nanoprobes to diagnose liver fibrosis (Sheng et al., 2018).
The wide-range composition, size, shape and changeable surface properties of NPs makes them unique, as controlled drug release, high divergence, extended bioavailability, reduction of toxic side effects and improvement of the pharmacokinetics of drugs (Petros et al., 2010). An interesting and beneficial significance of nano systems is the possibility to diagnose and treat at the same time (Nagorniewicz et al., 2019).

Human diseases and Nanotechnology
Medical NPs are designed nano-structures from drugs, peptides, proteins, or nucleic acids and are loaded in carriers. Hereafter, NPs are used to resolve the conflict of barriers that conquer the delivery of drugs and imaging labels to targets. These barriers may be attributed to biological, biophysical, and biomedical problems. Transforming the drugs and genes into NPs to target specific cells or tissues via coating or attaching ligands, may alter the therapeutic hypothesis, thus therapeutic agents can reach directly the needed site.
There are marked benefits to use NPs delivery systems as: (1) Avoiding the degradation of the therapeutic agent by being inactivated or being broke down until it scopes the site of action; (2) optimizing the pharmacological effects that result in enhancing drugs' bioavailability (3) possibility of integration of hydrophobic and hydrophilic molecules; (4) reduction of drug blood level fluctuations that may subject patients to lower risk of noneffective or toxic doses (5) decreasing the side effects and toxicity of drugs (6) controlled drug release that is needed in many pathological conditions; (7) using different routes of administration (8) due to the high affinity between the site of interest and the nano drug, active targeting is fulfilled Liver and spleen are considered to be the chief organs of accumulation of NPs (Moghimi et al., 2001) owing to help them reach the blood and high number of tissue-resident phagocytic cell (Giannitrapani et al., 2014;Mohammadpour et al., 2020).

Common NPs types recruited to treat liver diseases
The NPs used in drug delivery and biomedical studies is promptly increasing. NPs can be categorized into two main categories: inorganic and organic NPs and inorganic NPs have outstanding properties that made them of great interest.
Generally, inorganic NPs constitute a metal oxide as titanium oxide and iron oxide, or metal central core with a surface protective organic layer. This outer layer plays a crucial role in preventing the degradation of the inside content and permitting the pairing of biomolecules with the reactive groups as thiols and amines in order to link proteins, peptides, and folic acid.
Inorganic NPs have significant attention in recent years as they possess many physicochemical properties which are attributed to their different size and material, as compared to organic NPs. They possess unique magnetic, electronic, and optical properties which can be customized by regulating the size, composition, structure, and shape. Due to their inertness, easy functionalization, and stability, they have been considered as an attractive alternative over organic NPs for imaging specific tissues and drug delivery (Ketabat et al., 2019).
Organic Polymeric NPs include numerous sorts of nanoparticles for example; liposomes, solid lipid nanoparticles (SLN), and nanostructured lipid carriers (NLCs) (Figure 1). These biocompatible and biodegradable polymeric NPs are now of great importance as possible drug delivery preparations which can be used as carriers of DNA in gene therapy, administered in drug targeting to particular organs or tissues, and they can deliver peptides and proteins orally.
The US Food and Drug Administration had approved PLA and PLGA biodegradable polymeric nanoparticles. Liposomes are synthetic sphere-shaped vesicles that contains one or more phospholipid bilayers enfolding an aqueous compartment. A variety of hydrophobic and hydrophilic medications can be encapsulated in the dual compartment structure of liposomes where hydrophilic drugs inside the internal aqueous core shaped by the lipid membrane, while hydrophobic drugs can be fused into the bilayer.
Liposomal NPs are the simplest form of NPs that have several advantages as good biocompatibility, easy preparation, increased uptake and reduced systemic toxicity (Datta et al., 2020;Wang et al., 2020a).
Conventional liposomes are immediately removed from the blood circulation and that is rendered to their high affinity for the reticuloendothelial system (RES), that is why they are coated with hydrophilic molecules linked to the liposomal formulation by a lipid anchor so as to prolong liposome circulation over time (Immordino et al., 2006). Consequently, the pharmacological potency has been improved as well as reduction in the used dose.
Solid lipid nanoparticles (SLNs), were introduced in the 1990s (Muller et al., 2000) and they were considered a new carrier system other than liposomes, polymeric nanoparticles and emulsions.
The solid lipid core can include triglycerides, fatty acids, steroids, waxes and glyceride mixtures. SLNs have advantages over the traditional systems as it avoids some of the disadvantages of other types since they are produced easily without the usage of organic solvents, and they can be synthesized on a large scale at low cost (Bondi et al., 2003). Moreover, they do not cause biodegradability problems or toxicity (Puri et al., 2009;He et al., 2020).
NLCs were introduced by late 1990s to improve the capabilities of other previous

Potential Targets Of Liver Fibrosis
Choosing the target and the carrier while targeting hepatic ailments should be conferring to the type of the ailment. The hepatoprotective drugs should be directed at the liver cells, while in autoimmune and inflammatory conditions, drugs should be delivered to the Kupffer cells. Moreover, antifibrotic drugs perform their action via targeting the hepatic stellate cell. Erroneous drugs uptake lead to ineffectiveness as well as possible detrimental effects.
Triggered HSCs primarily elaborate during inflammation, angiogenesis, and fibrogenesis responses for the progress of liver fibrosis. Approaches to target HSCs may potentially treat liver fibrosis due to their involvement in the development of the disease. Another strategy includes using anti-inflammatory drugs or collagen deposition inhibitors.

Hepatic stellate cells (HSCs)
Primary modulations in the genetic expression as well as phenotypic changes in HSCs are the initial stage in the development of liver fibrosis. These modulations and changes are rendered to cytokine and chemokine stimulation ( Figure 2).
Cytokines secreted by Kupffer cells play a chief role in the activation of HSCs (Pradere et al., 2013).
When HSCs become activated, they release reactive oxygen species (ROS), NF-κβ and damage-associated molecular patterns (DAMP) which trigger the innate immune response. Thus, leukocytes release lipid peroxides and TNF-related apoptosis inducing ligand (TRAIL) and other ligands (Lan et al., 2020). The activation of the HSCs, results in matrix synthesis, proliferation, and loss of retinoids and all these changes ends by liver fibrosis. As an alternative of causing injury to normal liver cells, those activators enhance the inactive HSCs into activated form. Liver damage induced by carbon tetrachloride in rodent stimulates the HSC and liver fibrosis mechanism (Miao et al., 2019). TGF-β and PDGF are two main cytokines that lead to HSC activation followed by activation of the cells during the progression of liver fibrosis.

Abbreviations
"NO, nitric oxide; HSC, hepatic stellate cell; CTGF, connective tissue growth factor; TGF-β, transforming growth factor β; PDGF, plateletderived growth factor; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase" Transforming Growth Factor Beta (TGF-β) Transforming Growth Factor Beta (TGF-β) is: "one of the most potent cytokines to induce fibrogenesis as it activates HSCs". It has been found that different miRNAs are indulged in the events of liver fibrosis and they control many critical steps in its development. From the intracellular pivotal effectors that has been discussed in affecting TGF-β are the SMAD proteins. (Hellerbrand et al., 1999;Li et al., 2018).
Concerning liver fibrosis SMAD2 and SMAD7 are hepatoprotective while SMAD3 and SMAD4 are profibrotic proteins. The interference in the pathway of SMAD3 blocks the epithelial myofibroblast transition and stops the expression of collagen type 1. Moreover, SMAD4 enhances the responsive promoter effect of SMAD3 resulting in intensifying liver fibrosis.
On the other hand SMAD7 inhibits SMAD3 fibrogenesis so it is an evidence now that various miRNAs participate in the hepatic fibrotic disease and hence targeting TGF-β/SMAD signaling could be an attractive crucial target in alleviating liver fibrosis (Zhang et al., 2020) ( Figure 3).
The mitogen-activated protein kinase (MAPK) pathway is also affected by the TGF-β, with extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) changes (Hanafusa et al., 1999). Alpha smooth muscle actin (a-SMA), proteoglycans, as well as collagen with its two types; types I and III are upregulated through induced transcription after their activation (Breitkopf et al., 2006).
The level of both a-SMA as well as the levels of collagen I and III are from the important markers that indicate HSCs activation and the degree of liver fibrosis so as to follow up and monitor the case. The elevation of such markers indicates the aggravation of the liver fibrosis (Tomasek et al., 2002;Schuster-Gaul et al., 2020).
Cells can receive external signals and respond appropriately via the MAPK pathway mainly throughout the epithelial-mesenchymal transition (EMT) (Gui et al., 2012) EMT is an important process during the development of the embryo, fibrosis, and tumor progression in which the epithelial cells attain mesenchymal, fibroblastlike properties and display low intercellular adhesion and increased motility.
TGF-b signaling is the preliminary pathway to trigger EMT, and its relationship with the Smad family is well considered. Previous studied showed that the MAPK and TGF-b signaling pathways relate together and exert a synergistic effect on the release of growth factors and cytokines that endorse EMT" (Gui et al., 2012).

Platelet-Derived Growth Factor (PDGF)
From the most important roles of mitogen PDGF and its receptor PDGF receptor-b (PDGFRb) is acting on HSCs leading to their proliferation and migration (Wong et al., 1994;Kostallari et al., 2018).
Patients suffering from chronic liver diseases can be monitored and followed up by checking the inflammatory extent of their case which is a reflection and it is linked to the expression of PDGF (Zuo et al., 2019). It I to be noted that in spite of the expression of PDGFRb mRNA expression is found to be in both quiescent and activated HSCs, only protein production was limited to the activated HSCs cells (Henderson et al., 2013).

Vascular Endothelial Growth Factor (VEGF)
The vascular endothelial growth factor (VEGF) was documented as vascular permeability factor that is formed by fibroblasts and it is wellthought as a signal protein responsible for the initiation and stimulation of blood vessels. They are involved with other mediators in both the de novo formation of the embryonic circulatory system and angiogenesis (Yang et al., 2014).
In liver fibrogenesis, VEGF stimulates the cell proliferation of HSCs, which includes angiogenesis in the distorted liver tissue. VEGF has a multifaceted role in fibrogenesis, liver tissue repair and reversal of fibrosis (Kantari-Mimoun et al., 2015). VEGF also controls the migration of monocytes, penetrability of liver sinusoidal and scar-associated macrophage function, which are considered a fibrotic resolution and tissue repair processes (Yang et al., 2014).

Connective Tissue Growth Factor (CTGF)
Huang et al., 2012 stated that "the connective tissue growth factor (CTGF) is markedly expressed in the liver suffering from fibrosis when it is compared to the normal liver and that it is a potent fibrogenic cytokine that is similar to PDGF and it contributes to the accumulation of ECM resulting in a series of hepatic fibrogenic actions" (Huang et al., 2012). CTGF sensitizes HSCs that mainly produces it. CTGF is considered to be one of the main critical effectors that pushes the production of collagen and its expression is associated with the severity and degree of liver fibrosis (Chen et    These NPs also possess an anti-migratory and anti-adhesive effects via regulating gene expression of the epithelial mesenchymal transition (EMT) and work on the reversal of TGF-bactivated HSCs to the inactive state ( Figure 4). The anti-inflammatory effect of Cerium oxide NPs causes the decrease of liver steatosis, liver fibrosis and portal hypertension (Oro et al., 2016). Oral administration of trimanganese tetraoxide Mn3O4 NPs showed an mitigation of CCl4 induced liver fibrosis (Adhikari et al., 2016).
Some studies indicated that Zinc oxide (ZnO) NPs reduced liver fibrosis induced by dimethylnitrosamine through more than one mechanism, namely, reducing oxidative stress, lipid peroxidation, and inflammation (Rani et al., 2018).
Further types of inorganic NPs are used to ameliorate liver fibrosis other than metal oxide NPs. Gold NPs reduced liver fibrosis methamphetamineand ethanol-induced liver injury in rats via hindering Kupffer cells and HSCs activities (de Carvalho et al., 2018). This mechanism includes the regulation of phosphatidylinositol 3-kinase/ Akt (AKT/PI3K) and MAPK signaling pathways by gold NPs, thus reducing both pro-inflammatory cytokine release and oxidative stress. Besides, Bai et al., 2020 stated that "vitamin E-modified selenium NPs can reduce liver fibrosis by ameliorating of the oxidative stress" (Bai et al.,  2020a). It is prominent that the dynamic targeting is imperative for the targeting the distorted tissues as well as avoiding the normal tissues, thus, increasing the efficacy of these drugs and limiting their detrimental effects. Hence, "Active targeting is a way to increase the quantity of drug brought to the target cell when compared to free drug or nano drugs passively targeted".

NPs as Drug Transporters without Targeting Ligand for the Treatment of Liver Fibrosis
The liver is the central metabolic and excretory organ in the body and NPs are used as drug carriers due to their dimensions which can accumulate and target liver cells for the treatment of liver fibrosis.

Lipid-based NPs
Lipid-based NPs are the most significant vehicles due to their good biocompatibility and low toxicity (Bottger et al., 2020). Reebye et al., 2018 reported that: "CCAAT/enhancer-binding protein alpha (CEBPA), a master transcriptional factor in the liver can reduce fibrosis and restore liver function via resetting the hepatocyte natural gene regulatory mechanism. They also stated that the small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposome NPs can upregulate CEBPA expression, thus ameliorating the fibrosis" (Reebye et al., 2018).
In another study, authors reported a depletion in inflammatory cytokines, a-SMA and collagen after administration of

Polymer-based NPs
Polymer-based NPs have been used as drug carriers for liver fibrosis treatment. Leber et al., 2017, mentioned that the ketal cross-linked cationic nanohydrogel particles were synthesized to deliver Cy5-labeled anti-col1á1 siRNA, leading to PLGA and eudragit were used as drug carriers. Phyllanthin was carried by PLGA to reduce the biochemical markers of liver fibrosis and the researchers found that it significantly depleted various liver enzymes mainly, aspartate aminotransferase and alanine aminotransferase and much more interesting, was the reduction in the The SAB@MSNs-RhB formulation revealed improved cellular uptake, enhanced efficacy and continual drug release in hepatic fibrosis. Mesoporous silica NPs reduces the secretion of inflammatory cytokines via inhibition of TnC expression, as well as hepatocyte migration (Vivero-Escoto et al., 2019). Hesperetin loaded on PEGylated gold NPs showed significan antioxidant, anti-inflammatory and antiproliferative when compared to hesperetin alone in hepatocarcinogenesis induced by diethylnitrosamine in rats (Krishnan et al., 2017).

Protein-based NPs
Due to the biocompatibility and low immunogenicity of protein-based NPs, they were the most suitable drug carriers to be studied in the Liver fibrosis is attenuated using free dexamethasone treatment. Human serum albumindexamethasone NPs have been developed to transport dexamethasone to the cells playing a crucial role in the pathogenesis of liver fibrosis, namely non-parenchymal hepatic cells. Such modification leads to significant inhibition of TNF-a that attenuated liver fibrosis (Melgert et  al., 2000).

NPs as Drug Transporters with Targeting Ligands for the Treatment of Liver Fibrosis
Actually, the random drug delivery systems used in attenuating liver fibrosis are of low clinical effectiveness specially when compared to the target designed ones. The most efficient systems that can be used as a way to deliver drugs to a certain place are the nano formulations. Many studies in recent years showed advanced way of using nanoparticles in targeting hepatic fibrotic cells to alleviate liver fibrosis. It is to be noted as a rule that: "various receptor types that are expressed/ over-expressed on HSCs have been targeted using the suitable targeting ligands that are conjugated at "A drug-carrier construct basically consists of at least three elements: a carrier (or core molecule), a homing device, and a drug". Targeting high-affinity membrane receptor for retinol binding protein (rRBP) HSCs, precisely being retinoid storing cells in the liver, express retinol binding protein (rRBP) receptors that plays an important role in binding and taking-up vitamin A (VA). Thus, vitamin A-conjugated liposomes that are loaded with "siRNA against gp46 (the rat homolog of human heat shock protein 47)" that is involved in inhibition of collagen secretion, were injected intravenously into the fibrotic livers in rats.
Extensive decrease of collagen secretion and lessening in fibrosis was revealed after such drug treatment. It is to be noted that in normal A study used retinol binding protein receptor and "collagenase I co-decorated polymeric micelles" based on "PLGA-b-poly (ethylene glycol)-maleimide (PLGA-PEG-Mal)" (named CRM) as HSC-targeting nanodrug delivery systems for treatment of liver fibrosis (Fan et al., 2020). In this experiment, the decoration of collagenase I enabled the nanocarrier penetration to the fibrotic tissue. In parallel to this outcome, CRMs potently degraded pericellular collagen I and successfully accumulated in the fibrotic liver hence, targeting activated HSCs (Fan et al., 2020).
A second-generation tyrosine kinase inhibitor, nilotinib (NIL) was loaded on CRM forming CRM/NIL. Such formulation was administered for the treatment of liver fibrosis and it exhibited outstanding antifibrotic effect (Fan et al., 2020) (Figure 7).
In addition, polymeric micelles (PVMs) designed with PLGA-polyspermine-PEG-VA were prepared to target HSCs and transport the silibinin and genetic drug siCol1a1 to the hepatic fibrotic tissue (Qiao et al., 2018). It significantly depleted collagen I and attenuated liver fibrosis (Luo et al., 2019). Other than polymeric micelles, other polymeric nano formulations have been designed for drug, nucleic acid and other therapeutic moieties delivery to particular sites for the treatment of liver fibrosis.
Hassan et al. reported that "chitosan NPs loaded with atorvastatin and JQ1 (a thienotriazolodiazepine) could ameliorate the cytokine-induced activation of HSCs and hence, reverse fibrotic response in rodent models and further conjugated with retinol leading to targeting and preventing HSC activation" (Hassan et al.,  2019).

Targeting retinoic acid receptor
Chondroitin sulfate micelles that are coupled with retinoic acid and doxorubicin had a specific action as they were taken up by activated HSCs selectively and not by normal hepatocytes (Luo et al., 2019). These micelles specifically gathered in the Golgi apparatus and triggered the destruction of the Golgi structure, as a result, collagen I production was depleted and this manifested antifibrotic effects in fibrotic rats (Luo et al., 2019).

Targeting PDGFRb on HSCs
A study was accomplished to explore the effects of a new TRAIL (TNF related apoptosis inducing ligand) formula that can target activated HSCs to alleviate liver fibrosis and explore the underlying mechanism.
Li et al., in 2020 stated that: "Plateletderived growth factor receptor â (PDGFRb) was co-expressed with TRAIL receptor 2 (DR5) in the activated HSCs and that the ZPDGFRb affibody with high affinity for PDGFRb could bind the activated HSCs and, thus, accumulate in the fibrotic liver ( Figure 8).
ZPDGFRb was fused to hTRAIL to produce the fusion protein Z-hTRAIL. Compared to hTRAIL, Z-hTRAIL showed greater in vitro cell binding and apoptosis-induction in aHSCs. In addition, Z-hTRAIL induced apoptosis of activated HSCs but spared other normal liver cells. In vivo, Z-hTRAIL accumulated preferentially in fibrotic livers and exerted greater effects than hTRAIL in inducing activated HSCs apoptosis and reducing extracellular matrix (ECM) deposition. These results demonstrated that the antihepatofibrotic effect of hTRAIL was improved by PDGFRbtargeted delivery (Li et al., 2020)".
The cyclic peptide pPB can predominantly recognize PDGFRb on the HSCs surface. Li et al., 2019 stated that pPB-modified liposomes was administered as a way to deliver recombinant human tumor necrosis factor-related apoptosisinducing ligand (rhTRAIL) to the activated HSC membrane, showed prolonging rhTRAIL presence and circulation in vivo and hence, alleviating fibrosis" (Li et al., 2019a).
Recently, pPB coupled to albumin was used to deliver the Rho-kinase inhibitor Y27632 to HSC leading to beneficial effects in vivo (Klein  et al., 2019). The same peptide was used to deliver lipid nanoparticles containing siRNA to HSC (Jia et al., 2018). In addition, a cyclic-RGD-based peptide was coupled to liposomes for the delivery of the hedgehog inhibitor vismodegib (Li et al., 2019b). This latter construct was quite effective in reducing fibrosis in two experimental animal models of liver fibrosis in mice".

Targeting CXCR4
"In recent years, another receptor recognizing peptide binding to the CXCR4 receptor has been developed (Sung et al., 2018) which was coupled to polymeric biodegradable nanoparticles to create a carrier that allowed the simultaneous delivery of sorafenib and the MEK inhibitor selumetinib to HSC (Sung et al., 2018).
This combination therapy displayed significant antifibrotic effects in a mouse model. The CXCR4 receptor was also used for the targeting of pirfenidone to HSC (Ullah et al., 2019). In this latter case however, the nanoparticles (liposomes) were not functionalized with receptor recognizing peptides but with the CXCR4 antagonist AMD3100 (plerixafor). The effect of pirfenidone, one of the few FDA-approved drugs against fibrosis, combined with blocking of the chemokine receptor CXCR4 may lead to synergistic effects and is another example of a successful combination therapy".
Vascular endothelial growth factor (VEGF) is a critical signaling protein that aids in the development of new blood vessels. VEGF participate in the mechanism that returns the blood supply to cells and tissues after being poorly oxygenated blood (Cameron et al., 2020).
It had been proven that in rat liver fibrosis models, treatment with drugs that neutralize VEGF produced by hepatocytes, can markedly alleviate liver fibrosis (Siddiqui et al., 2020). Liu et al., stated that "AMD3100-conjugated liposomes successfully delivered therapeutic VEGF siRNAs to activate CXCR4-overexpressed HSCs. The nano formulations interestingly, downregulated the expression of VEGF, decreased the mean vessel density, and normalized the hepatic vascular structure in the mice livers suffering from CCl4induced liver fibrosis. In addition, AMD3100 conjugated liposomes suppressed the proliferation and activation of HSCs showing antifibrotic effects". Targeting mannose 6-phosphate (M6P)/insulinlike growth factor-II receptor "Other than peptides and a receptor antagonist, sugar moieties have been used as homing devices as well" (Figure 8).
In fibrotic liver, mannose 6-phosphate (M6P)/insulin-like growth factor-II receptor is overexpressed in HSCs rendering it as an attractive targeting issue. Pairing of M6P-modified albumin to hesperidin-loaded liposomes showed marked improvement in the efficacy of chemical drugs and attenuated liver fibrosis (Morsy et al., 2018).

Targeting phosphatidylserine (PS)
Wang et al. concluded that: "Scavenger receptors expressed on liver endothelial cells and Kupffer cells have also been targeted using nano formulations. For instance, phosphatidylserine (PS), which acts as a specific recognition signal for the phagocytosis of apoptotic cells, can target macrophages. Wang et al. showed that PS-modified lipid carriers containing curcumin (Cur-mNLCs) exhibited enhanced retention time, bioavailability, and delivery effeciency of payload, as well as reduced liver damage and fibrosis in vivo" .

Targeting Integrins
Integrins are main receptor that clings the extracellular matrix proteins as laminins, collagens as well as fibronectin. Type VI collagen is dispersed in HSCs and they express integrins. Hence, targeting collagen VI integrins plays an important role in alleviating hepatic fibrosis (Beljaars et al., 2000). Du et al., 2007 suggested that: "RGDcoupled liposomes have efficiently delivered the encapsulated interferon (IFN)a-1 to the fibrotic liver in rat, achieving a considerable anti-fibrotic activity" (Du et al., 2007). Other studies followed the same path to alleviate different fibrotic tissues (Chung et al., 2016).

Targeting galactosyl receptor
The galactosyl receptor that is expressed on the hepatocytes plays a role in the internalization of molecular asialoglycoproteins and small particles. Knowing that, liposomes decorated with "p-aminophenyl a-D-galactopyranoside", which binds to galactosyl receptor, are used as transporters to target HSCs with quercetin.
Such "quercetin-loaded galactosylated liposomes" were accumulated 1.7 fold more in the rat liver than non-galactosylated liposomal quercetin and 3.4-fold more than the free quercetin leading to considerable hepatoprotection as it markedly prevented the depletion of native antioxidant levels and it inhibited the collagen production that is responsible for fibrogenesis as well (Mandal et al., 2007). A recent study too used Galactosyl Nanoparticles to Target HSCs Hepatocellular Carcinoma (Liao et al., 2021).

Nanomedicine In Liver Fibrosis Theranostics
"Theranostics", a hybrid word of "therapeutics" and "diagnostics", is accomplished by integrating diagnostic and therapeutic functions into a single nanoplatform. Theranostics has been presented as a new and revolutionary therapeutic perception in several types of diseases as liver fibrosis (Gou et al., 2018).
This new path allows concurrent diagnosis and treatment response by using tailored medicine with high specificity and accuracy.
One of the hallmarks of activated HSC is their increased expression of integrin avb3 on their surface (Schuppan et al., 2018). In one study, Zhang et al., 2019 stated that: "Hepatitis B core protein nanocages coated with RGD-targeting ligands overloaded with querecitin (RGD-HBc/ QR) showed selectivity to activated HSCs by targeting integrin ávâ3 which efficiently repressed the proliferation and activation of HSCs  and that by encapsulating a quercetingadolinium complex and/or labeling it with near infrared (NIR) fluorescent probes (Cy5.5), the resulted nano formulations (RGD-HBc/QGd) showed great potential as (Magnetic resonance imaging) MRI contrast agents and NIR fluorescent agents for liver fibrosis diagnosis in vivo".
Another study reported that "relaxinconjugated PEGylated superparamagnetic iron oxide NPs" (RLX-SPIONs) exhibited specific binding and uptake in TGF-b-activated HSCs, as well as strongly lessened cirrhosis and showed improved contrast in MRI (Nagorniewicz et al., 2019). Micelles attached with inorganic agents were also established for theranostics to alleviate liver fibrosis (Liu et al., 2021).
"Next to small chemical entities with antifibrotic effects, that have been coupled and incorporated in many of the above reviewed drug carriers, biological products like antisense oligonucleotides, siRNA, miRNA, and cytokines can be powerful tools to modify fibrosis. The therapeutic applications of siRNA have become clear in recent years, and many different miRNA and siRNA molecules (Omar et al., 2018) have been tested in experimental animal models of fibrosis. Delivery is the key issue for siRNA and miRNA molecules. Although cytokines play a key role in vivo in regulating fibrogenesis and are active in the picomolar range, their pleiotropic activities have strongly limited their therapeutical use. This may be solved using targeting strategies (Bansal et al., 2014)".

Antifibrotic Agents
Scientific communities are now focusing on the studies related to natural nano bioactive compounds to produce pioneering efficient active ingredients with minor side effects than synthetic prevailing molecules.
Application of nanotechnology systems using natural components in the medical field has been widely considered in the last few years (Ramana et al., 2014;Shah et al., 2020;Dehghani Tafti et al., 2021). There is evidence that the association of nono systems with natural compounds may help to reduce the progress of drug resistance and hence these systems play an important role in developing formulations that are target specific, potent and of little side effects for the treatment of liver fibrosis. It is to be renowned that natural components can either be used as the polymer which is the drug carrier (Figure 9) or as the therapeutic component.

Egyptian Experience
Various Egyptian research groups are involved in the advancement of nanocarriers for applications of drug-delivery including the usage of nanocarriers to improve the effectiveness of different pharmaceutical formulations including oral, nasal, parenteral, transdermal.. etc.
Hussein et al., 2017 compared the therapeutic potential of nano-encapsulated and nano-emulsion of a natural constituent, carvacrol, on thioacetamide-induced liver fibrosis in rats. The nano formulations of carvacrol succeeded to ameliorate thioacetamide-induced liver fibrosis (Hussein et al., 2017).
Khattab et al., developed and characterized self-nanoemulsifying formulation of coenzyeme Q10 (CoQ10) to investigate its biochemical and physiological effect on liver cirrhosis in CoQ10 rats and they compared it with CoQ10 powder. The team demonstrated that CoQ10 nano formulation attenuated thioacetamide-induced liver fibrosis mainly through inhibiting collagen production .
El-Mezayen et al., 2017 prepared a novel vitamin A-coupled imatinib-loaded liposomes regarding VA-coupling efficiency as well as imatinib entrapment resulting in significant antifibrotic effects with reduced cytotoxicity compared to conventional imatinib (El-Mezayen et al., 2017).
El-Mezayen et al., 2018 stated that: "vitamin A stores in hepatic stellate cells were targeted to deliver valsartan to HSCs to treat liver fibrosis and encapsulation of valsartan into vitamin A-coupled liposomes targeted HSCs, caused a significant activation of hepatic nuclear PPAR-g receptors in HSCs confirming the powerful antifibrotic activity of valsartan liposomes" (El-Mezayen et al., 2018).
Infection by Schistosoma mansoni is an endemic pathogen in Egypt and it is often progressed to severe hepatic disorders as liver fibrosis or necrosis. Two studies have been done depending on the improvement of the efficiency of praziquantel through nanotechnology ideas.
The first team intended to elevate the therapeutic outcome of the main antischistosomal drug worldwide, praziquantel through including it into a novel carrier which is solid lipid nanoparticles (SLNs). Such modulation enhanced praziquantel bioavailability and antischistosomal efficacy and hence alleviated liver fibrosis which is a common result of such case (Radwan et al., 2019).
The second Egyptian team of researchers intended to study the anti-schistosomal activity of curcumin as well as "curcumin loaded goldnanoparticles (Cur-GNPs)" in the presence of praziquantel. They found that the curcumin in combination with praziquantel altered the biochemical, and immunological damage and hence, alleviated liver fibrosis (Mokbel et al., 2020).

Conclusions and future perspectives
This review outlines the nanotechnology strategies that are used to progress novel specific and accurate systems for alleviating liver fibrosis. This field of nanomedicine systems continues to be increasingly growing and developing so as to aid in both the diagnosis and treatment of liver fibrosis.
Variable categories of inorganic and organic NPs have been widely inspected, including "metal oxide NPs, liposomes, metal NPs, protein NPs, polymer NPs and organic-inorganic hybrid NPs". Each category has its rewards and drawbacks. However inorganic NPs are found to be efficient with comparatively low synthesizing expenses, but their functionality and design elasticity are limited. On the other hand, organic NPs have wide functionalities as well as broad design flexibility but they are unstable and they are of high cost.
Many studies in recent years showed advanced way of using nanoparticles in targeting hepatic fibrotic cells to alleviate liver fibrosis as numerous receptor types that are expressed on HSCs have been targeted by means of the suitable targeting ligands that are conjugated at the surface of liposomes Recommendations to be considered to advance the medical application of nanomedicine systems in the future: 1. Developing stimuli-responsive nano systems, that can smartly respond to both endogenous or exogenous stimuli and release the needed therapeutic load at targeting sites. 2. Exploring advanced targeted therapies is a priority. 3. Employing clever nanomedicine structures that syndicate multiple functionalities, including prolonged blood retention, targeted delivery, responsiveness to stimuli, improved tissue penetration and disease progressive monitoring. 4. Regular assessment of long-term toxicity, p h a r m a c o k i n e t i c s o f t h e s y s t e m s a n d immunogenicity.