*4.3. NPs as Drug Carriers with Targeting Ligands for the Treatment of Liver Fibrosis*

Non-specific drug disposition limits the effective clinical use of traditional anti-fibrotic drugs. Targeting drug delivery to the fibrotic region can thus far be achieved using nanoformulations. As the sole hepatic VA storage cells with a crucial role in liver fibrosis, HSCs have been actively targeted by conjugating NPs with VA.

Liposomes loaded with drugs and HSC targeting components have been developed to target HSCs for the treatment of liver fibrosis (Table 2). In one study, VA-coupled liposomes were prepared to deliver imatinib. The hepatic accumulation of imatinib increased by about 13.5-fold, compared with imatinib treatment alone [88]. The nanoformulations not only inhibited the expression of phosphorylated PDGFR-β but also reduced the expression of profibrotic mediators such as hydroxyproline, TGF-β, and MMP2 with fewer adverse effects. In another study, VA-coupled liposomes were used to deliver valsartan, an angiotensin II receptor antagonist [89]. The nanoformulations increased the expression of hepatic Mas-receptor and PPAR-γ and potently normalized the level of fibrogenic mediators by improving the permeability and efficacy of valsartan.



Abbreviations: VA, A; TRAIL, apoptosis-inducing ligand; HSC, hepatic cell; CTGF, growth factor; MMP, metalloproteinase; NO, nitric oxide; VEGF, vascular endothelial growth factor.

Polymer-based NPs have also been fabricated to target HSCs by coupling with VA for liver fibrosis therapy (Table 2). An article reported on the preparation of retinol and collagenase I co-decorated polymeric micelles (CRM) based on PLGA-b-poly(ethylene glycol)-maleimide (PLGA-PEG-Mal) to be used as HSC-targeting nanodrug delivery systems for liver fibrosis therapy [100]. In the current study, the decoration of collagenase I could facilitate the nanocarrier penetration of the fibrotic liver. Consistent with this finding, CRMs were found to efficiently degrade pericellular collagen I and exhibit excellent accumulation in the fibrotic liver and accurate targeting of activated HSCs in a mouse hepatic fibrosis model. CRM/NIL loaded with nilotinib (NIL), a second-generation tyrosine kinase inhibitor used for the treatment of liver fibrosis, showed excellent antifibrotic efficiency (Figure 4). In addition, polymeric micelles (PVMs) formed with PLGA-polyspermine-PEG-VA were used to target HSCs and deliver the chemical drug silibinin and genetic drug siCol1α1 to the liver fibrosis site [101]. The double-loaded polymer micelle more efficiently reduced collagen I and ameliorated liver fibrosis, compared with the PVMS loaded with either the chemical drug only or genetic drug only. Chondroitin sulfate micelles coupled with retinoic acid and doxorubicin (DOX) (DOX + RA–CS micelles) were selectively taken up in activated HSCs and hepatoma cells, but not in normal hepatocytes (LO2) [102]. DOX + RA–CS micelles preferentially accumulated in the Golgi apparatus, destroyed the Golgi structure, and ultimately downregulated collagen I production in vitro and exerted synergistic antifibrotic effects on CCl4-induced fibrotic rat models.

**Figure 4.** Extracellular matrix-penetrating polymeric micelles for liver fibrosis therapy. (**A**) Schematic illustration of the preparation of four different polymeric micelles. (**B**) Schematic illustration of the proposed destiny of the four different polymeric micelles in vivo. The CRM/NIL is able to penetrate the collagen barrier and target activated HSCs. Internalization of CRM/NIL allows the release of NIL, which reduces expression of the metallopeptidase inhibitor, TIMP-1, which in turn enhances collagen I degradation, thereby exerting therapeutic action against liver fibrosis. Reproduced with permission from [100]. Copyright Elsevier, 2020.

Apart from polymeric micelles, other polymer nanoformulations have also been constructed for the delivery of drugs, nucleic acid and other therapeutic moieties for the treatment of liver fibrosis (Table 2). In one study, retinol-conjugated polyetherimine NPs adsorbed plasma proteins, particularly retinol-binding protein 4 (RBP), forming a protein-coated complex [103]. The adsorbed RBP could direct the NPs into HSCs. After being loaded with antisense oligonucleotides, NPs effectively suppressed the expression of collagen I, consequently ameliorating hepatic fibrosis. Hassan et al. reported that

chitosan NPs loaded with JQ1 (a small molecule that could abrogate the cytokine-induced activation of HSCs and reverse fibrotic response in animal models) and atorvastatin and further conjugated with retinol could target and prevent HSC activation [105].

In addition to VA, cyclic peptide pPB can particularly recognize PDGFRβ on the surface of HSCs (Table 2). A study used pPB-modified liposomes to deliver recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) to the HSC membrane, prolonging rhTRAIL circulation in vivo and alleviating fibrosis both in vitro and in vivo [90]. Similarly, the CXCR4 antagonist AMD3100 could target HSCs [91]. AMD3100-conjugated liposomes efficiently delivered therapeutic VEGF siRNAs to activate CXCR4-overexpressed HSCs both in vitro and in vivo. The nanoformulations downregulated the expression of VEGF, reduced the mean vessel density, and normalized the hepatic vascular structure in the livers of mice with CCl4-induced liver fibrosis. Moreover, AMD3100 encapsulated in liposomes also exhibited antifibrotic effects by suppressing the proliferation and activation of HSCs. Mannose 6-phosphate (M6P)/insulin-like growth factor-II receptor, overexpressed in HSCs, was also used as the targeting site. Conjugation of M6P-modified albumin to hesperidin-loaded liposomes improved the efficacy of chemical drugs and attenuated liver fibrosis [92].

Hepatic macrophages play important roles in the pathogenesis of liver fibrosis and act as target sites for the treatment of liver fibrosis (Table 2). Scavenger receptors expressed on liver endothelial cells and Kupffer cells have also been targeted using nanoformulations. 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 efficiency of payload, as well as reduced liver damage and fibrosis in vivo [99].
