*4.2. NPs as Drug Carriers without Targeting Ligand for the Treatment of Liver Fibrosis*

The liver is the main metabolic and excretory organ in the body in which NPs can accumulate and accomplish passive targeting because of their size. Thus, NPs have been widely used as drug carriers for the treatment of liver fibrosis. Lipid-based NPs have been recognized as the most powerful vehicles because of their good biocompatibility and low toxicity (Table 1) [69]. CCAAT/enhancer-binding protein alpha (CEBPA), a master transcriptional factor in the liver, resets the natural gene regulatory mechanism of hepatocytes to reduce fibrosis and reverse liver dysfunction. Small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposome NPs can upregulate CEBPA, thereby reducing fibrosis [70]. After Cur-mNLCs treatment, pro-inflammatory cytokines, collagen fibers and α-SMA were reduced, while hepatocyte growth factors (HGF) and MMP2 were increased. Cationic lipid NPs loaded with small interfering RNA to the procollagen α1(I) gene (LNP-siCol1α1) can be retained in the liver of fibrotic mice and accumulate in nonparenchymal liver cells, specifically blocking procollagen α1(I) expression and inhibiting liver fibrosis progression without noticeable side effects [20].


**Table 1.** NPs as drug carriers without targeting ligands in liver fibrosis treatment.

Polymer-based NPs have been fabricated as drug carriers for the treatment of liver fibrosis (Table 1). In one study, ketal cross-linked cationic nanohydrogel particles were synthesized to deliver Cy5-labeled anti-col1α1 siRNA, which enhanced carrier and payload accumulation in the fibrotic tissue and prevented fibrosis progression [73]. PLGA and eudragit have also been employed as drug carriers. In one study, phyllanthin was carried by PLGA to reduce liver marker enzymes, namely alanine aminotransferase and aspartate aminotransferase, as well as collagen [74]. In another study, silymarin was delivered by eudragit NPs for the treatment of liver fibrosis by decreasing the expression of TNF-α, TGF-β1, TIMP-1, and CK-19. Moreover, nanoformulations were also found to increase HGF and MMP-2 expression and the MMP-2/TIMP-1 ratio [76].

In addition, inorganic NPs such as silica-based NPs were prepared as drug carriers because of their porous structures (Table 1). Salvianolic acid B (SAB) loaded rhodamine B covalently grafted mesoporous silica NPs (SAB@MSNs-RhB) were prepared for liver fibrosis therapy through the [77]. The SAB@MSNs-RhB formulation exhibited improved cellular uptake, sustained drug release, and enhanced efficacy in anti-ROS/hepatic fibrosis. Small interfering tenascin-C was delivered by mesoporous silica NPs to reduce the expression of TnC, an ECM glycoprotein, consequently reducing the secretion of inflammatory cytokines and hepatocyte migration [78]. Compared with hesperetin alone, hesperetin loaded on PEGylated gold NPs showed higher antioxidative, anti-inflammatory, anti-proliferative, and anti-fibrotic activities in diethylnitrosamine-induced hepatocarcinogenesis in rats [79]. Graphene nanostars conjugated with a PAMAM-G5 dendrimer were prepared for the selective targeting and delivery of a plasmid expressing collagenase metalloproteinase 9 under the CD11b promoter into inflammatory macrophages in cirrhotic livers. The nanoformulations promoted the macrophage switch from inflammatory M1 to proregenerative M2 and reduced selectively and locally the presence of collagen fibers in fibrotic tracts [83].

Protein-based NPs currently show great potential as drug carriers for the treatment of liver fibrosis because of their biocompatibility and low immunogenicity (Table 1) [87]. Algandaby et al. reported that curcumin-loaded zein nanospheres showed high efficiency in attenuating the hepatic gene expression of collagen I, the tissue inhibitor of MMP2, and TGF-β, as well as downregulating MMP2 expression [84]. Moreover, compared with free berberine, berberine entrapped in glucose-modified albumin NPs more efficiently inhibited the growth of the human hepatic stellate cell line LX-2 and reduced liver fibrosis in vivo [85]. Human serum albumin-dexamethasone NPs were also fabricated to deliver dexamethasone to non-parenchymal hepatic cells, which play an important role in the pathogenesis of liver fibrosis. This treatment efficiently inhibited TNF-α production, hence the significant decrease in fibrosis relative to that in rats treated with free dexamethasone treatment [22]. Another study reported on the preparation of avidin-nucleic-acid-nano-assemblies (ANANAS), which are NPs based on polyavidin. These NPs were generated from a nucleic acid filament and avidin, a protein in egg whites. These NPs were designed to selectively deliver dexamethasone to the liver, particularly to the liver immunocompetent cells, and thereby improve the therapeutic efficacy by reducing interlobular collagen I deposition and MMP13 [86].
