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Golgi Apparatus-Targeted Chondroitin-Modified Nanomicelles Suppress Hepatic Stellate Cell Activation for the Management of Liver Fibrosis Jingwen Luo, Pei Zhang, Ting Zhao, Mengdi Jia, Peng Yin, Wenhao Li, Zhi-Rong Zhang, Yao Fu, and Tao Gong ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06924 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Golgi Apparatus-Targeted Chondroitin-Modified Nanomicelles Suppress Hepatic Stellate Cell Activation for the Management of Liver Fibrosis

Jingwen Luo†, Pei Zhang†, Ting Zhao†, Mengdi Jia†, Peng Yin†, Wenhao Li†, Zhi-Rong Zhang†, Yao Fu†, and Tao Gong*†

†Key

Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry,

Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610064, China

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ABSTRACT: Liver fibrosis is a serious liver disease associated with high morbidity and mortality. The activation of hepatic stellate cells (HSCs) and the over production of extracellular matrix proteins are key features during disease progression. In this work, chondroitin sulfate nanomicelles (CSmicelles) were developed as a delivery system targeting HSCs for the treatment of liver fibrosis. CS-deoxycholic acid conjugates (CS-DOCA) was synthesized via amide bond formation. Next, retinoic acid (RA) and doxorubicin (DOX) were encapsulated into CSmicells to afford DOX+RA-CSmicelles co-delivery system. CSmicelles were selectively taken up in activated HSCs and hepatoma (HepG2) cells other than in normal hepatocytes (LO2), the internalization of which was proven to be mediated by CD44 receptors. Interestingly, DOX+RA-CSmicelles preferentially accumulated in Golgi apparatus, destroyed the Golgi structure, and ultimately downregulated collagen I production. Following tail-vein injection, DOX+RA-CSmicelles were delivered to the cirrhotic liver and showed synergistic antifibrosis effect in the CCl4-induced fibrotic rat model. Further, immunofluorescence staining of dissected liver tissues revealed CD44-specific delivery of CS derivatives to activated HSCs. Together, our results demonstrate the great potential of CS based carrier systems for the targeted treatment of chronic liver diseases.

KEYWORDS: chondroitin sulfate, liver fibrosis, Golgi apparatus, activated hepatic stellate cells, targeted delivery

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As one of the most important organs in the human body, liver is associated with various biological functions such as filtering blood, secreting bile for digestion and producing various proteins. Liver fibrosis occurs in multiple chronic liver diseases,1, 2 resulting from excessive accumulation of extracellular matrix proteins including types I and III collagen, proteoglycans, fibronectin, and hyaluronic acid.3, 4 Advanced liver fibrosis leads to cirrhosis, organ failure, and even hepatocellular carcinoma.5 The activation and proliferation of hepatic stellate cells (HSCs) are critical in the pathogenesis of liver fibrosis, which produce extracellular matrix proteins in response to the injury.6 Accordingly, activated HSCs become the targets of antifibrotic therapy. Recently, treatments using nanotechnology have attracted extensive attentions owing to the organ level targeted delivery of therapeutic cargos to the liver.7-9 For example, antisense oligonucleotide-laden retinol-conjugated polyetherimine particles achieved HSCs targeted delivery for the treatment of liver fibrosis.10 However, the lack of targeting precision, and the systemic toxicity limit the potential application and translation of these strategies.11 CD44 is specifically overexpressed on activated HSCs and various tumor cells.12, 13 Natural substrates that specifically interact with CD44 include hyaluronic acid (HA) and chondroitin sulfate (CS).14-16 From our preliminary studies, CS derivatives showed more enhanced uptake efficiency than HA derivatives (same molecular weight and same degree of substitution with CS derivatives) in activated HSCs indicating better affinity with CD44 (Supporting Figure 1). Thus, CS has been suggested as a suitable material candidate to fabricate delivery systems to achieve HSCs-specific drug delivery. As part of the extracellular matrix (ECM), CS is a linear polysaccharide with biocompatibility, biodegradability, and low immunogenicity.17-19 CS plays critical roles in the swelling and hydration of collagen fibrils, cell signaling, recognition and connection between ECM and cells.20 For example, CS-polyethylenimine conjugates were synthesized to enhance the transfection efficiency of pDNA in various tumor cell lines and to achieve targeting Ehrlich ascites tumor in mice.21 In our preliminary studies, authors investigated the subcellular localization of FITC labeled CS derivatives in activated HSCs. Interestingly, our results showed FITC labeled CS derivatives underwent endocytosis via CD44 receptor mediated internalization pathway and selectively accumulated in the Golgi apparatus. 3


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Golgi apparatus is responsible for processing proteins for secretion, containing a set of glycosylation enzymes that attach sugar monomers to proteins as the proteins move through the apparatus.22-24 Thus, strategies that destroy the structure and function of Golgi apparatus in activated HSCs may potentially inhibit collagen synthesis and benefit the treatment of liver fibrosis. Primary liver cancer is often accompanied with fibrosis, because hepatocellular carcinoma and fibrosis follow similar patterns of variation, and these two diseases share etiologic factors.25 In our previous work, low-dose doxorubicin (DOX, 0.5 mg/kg) reduced the degree of fibrosis while killing liver cancer cells in primary tumor bearing mice. Low-dose DOX presumably interfered with the function of HSCs. Greupink R. et al also concluded that low-dose DOX might be used to inhibit liver fibrosis.26, 27 Recently, retinoic acid (RA) has been shown to reduce HSCs proliferation and collagen production in culture.28, 29 Wang L. et al reported that RA suppressed liver fibrogenesis through inhibition of type I collagen production in liver and reduction of oxidative stress.30 Moreover, DOX and RA easily form DOX-RA complexes via charge interaction, and as a result, encapsulation efficiency of drug delivery system will be improved greatly. Thus, a combination of low-dose DOX with RA is proposed for the treatment of liver fibrosis. Here, we report a self-assembled nanomicelle system based on chondroitin sulfatedeoxycholic acid conjugate (CS-DOCA), phospholipid and sodium deoxycholate. The lipophilic side chains of CS-DOCA are inserted into the hydrophobic core of micelles via intermolecular hydrophobic interaction, while the hydrophilic main chains on the surface of the nanomicelles function as targeting ligands to reach activated HSCs (Figure 1). In the following study, the sub-cellular distribution and the internalization pathways of such nanomicelles were carefully investigated. A rat model of primary liver fibrosis was then established to evaluate the therapeutic efficacy of the DOX and RA combination strategy via systemic administration of CS based nanomicelles.

RESULTS AND DISCUSSIONS 4


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With rapid advances in nanomedicine, nanotechnology may allow therapeutic approaches for the targeted delivery of therapeutics in the treatment of liver diseases, including inflammation, fibrosis or cancer.31-33 Some polymers or polymeric drug conjugates bind with high affinity to specific selectins thus impacting hepatocyte selectivity.34, 35 Given the overexpression of CD44 receptors on activated HSCs during liver injury,36, 37 we explored using CS in the context of healthy and injured liver in rats. Even though cell-specific targeting delivery systems have been extensively studied in the field,38-40 organelle-specific delivery in liver associated cells has rarely been reported. The following study aims to explore the target ability of CS based nanomicelles (CSmicelles) for the Golgi apparatus-specific delivery to suppress HSCs and the therapeutic efficacy of DOX and RA combination for the management of liver fibrosis via CSmicelles in animal models.

Preparation and Characterization of CSmicelles. CSmicelles were synthesized via a thinfilm hydration method. Particle size, polydispersity index (PDI), and zeta potential (ζ) of blank CSmicelles, doxorubicin and retinoic acid loaded CSmicelles (DOX+RA-CSmicelles), doxorubicin loaded CSmicelles (DOX-CSmicelles), retinoic acid loaded CSmicelles (RACSmicelles), and doxorubicin and retinoic acid loaded micelles (without CS-DOCA, DOX+RA-micelles) were shown in Supporting Table 1. All formulations displayed uniform size distributions around 40 nm (Supporting Figure 2). All CSmicelles showed negative surface charges of around -20 mV, while unsubstituted micelles had surface zeta potentials of around 7.2 mV, possibly owing to the presence of anionic polymer CS on the surface of CSmicelles. Because of their structural characteristics, DOX-NH3+ and RA-COO- formed DOX-RA complexes easily. The oil-water partition coefficient of the DOX-RA complex was 1.22 whereas -3.37 for DOX·HCl and 3.70 for RA. The increase in lipid solubility for DOX benefited the encapsulation efficiency. The encapsulation efficiencies of DOX and RA in CSmicelles were increased to 95.8 ± 1.2% and 97.3 ± 1.7%, respectively, while only 56.1 ± 1.4% for DOX-CSmicelles. Total drug loadings of DOX and RA in DOX+RA-CSmicelles were around 3.3%. The critical micelle concentration (CMC) of CSmicelles was determined as 11.22 5


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± 1.01 μg/mL (Supporting Figure 3). Next, the stability of obtained micelles was investigated at 37 °C in bovine serum for 12 h. The size of micelles remained unchanged for up to 6 h in serum (Supporting Figure 4). Because the retention time of micelles in the blood was < 6 h, micelles were expected to maintain their structural stability in the blood circulation for at least 6 h. To evaluate the safety of CSmicelles, the hemolytic activity of CSmicelles was investigated, which showed no significant hemolytic activity even at the concentration of 10 g/L (Supporting Figure 5). Besides, DOX and RA showed sustained release from DOX+RA-CSmicelles lasting for up to 24 h in vitro (Supporting Figure 6). In the initial 4 h, the cumulative release percentage of free DOX reached 80%, while less than 20% of DOX release was observed for DOX+RACSmicelles. DOX+RA-CSmicelles and DOX+RA-micelles showed similar DOX release profiles, indicating that the presence of CS did not significantly affect DOX release. In comparison, RA released more slowly than DOX from each group, which can be explained by the high lipophilicity and slow dissolution rate of RA.

CSmicelles selectively deliver DOX and RA to cells with high levels of CD44 expression. CD44 is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and migration, which is expressed in a large number of mammalian cell types.41 Over expressions of CD44 were observed on the activated HSCs and HepG2 cells with the positive rates of 32.36% ± 6.27% and 26.61% ± 4.0%, respectively (Supporting Figure 7). Significant differences were observed in the positive rate between activated HSCs, HepG2, LO2, and human umbilical vein endothelial cells (HUVECs) (P < 0.05). Uptake and intracellular distribution of CSmicelles in above cells were evaluated by confocal microscopy. After incubating for 2 h, DOX fluorescence was observed primarily in the nuclei (Figure 2 A and Supporting Figure 8A). In comparison, a larger amount of DOX from CSmicelles could be taken by hepatoma cells and fibrosis-related cells than LO2 and HUVEC cells. Moreover, DOX+RA-CSmicelles showed the highest fluorescence intensity in HSCs and HepG2 cells, followed by DOX+RA-micelles and doxorubicin and retinoic acid solution (DOX+RA-solution) (Figure 2 A and Supporting 6


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Figure 8A). For LO2 and HUVEC, no significant differences in uptake efficiency were observed among three groups. To determine the uptake efficiency of DOX and RA, we performed uptake experiments using liquid chromatography-mass spectrometry (LC-MS) to analyze the concentrations of internalized DOX and RA in cell lysates. After 4 h incubation, HSCs took up substantially more DOX when exposed to DOX+RA-CSmicelles (11096 ng/mL) than to DOX+RA-micelles or DOX+RA-solution (7194 ng/mL, 4561 ng/mL; Figure 2B and Supporting Figure 8B). Similar trends were observed for RA and for HepG2 cells. In contrast, the uptake efficiencies of three formulations in LO2 cells (DOX+RA-CSmicelles, 4305 ng/mL; DOX+RA-micelles, 4410 ng/mL; DOX+RA-solution, 2713 ng/mL) and HUVECs (DOX+RA-CSmicelles, 1808 ng/mL; DOX+RA-micelles, 1892 ng/mL; DOX+RA-solution, 1392 ng/mL) remained relatively low. Overall, DOX+RA-CSmicelles significantly enhanced the uptake of DOX and RA in activated HSCs and HepG2 cells with high CD44 expression. More importantly, DOX+RA-CSmicelles showed enhanced cytotoxicity against activated HSCs and HepG2 cells compared to other micelles at the same dose (Figure 2C, Supporting Figure 8C). DOX+RA-CSmicelles showed lower IC50 on activated HSCs and HepG2 cells than on HUVECs and LO2 cells (Supporting Table 2). The modification of CS on micelles likely increased the affinity of micelles to the target cell membrane. In addition, blank CSmicelles displayed minimum cytotoxicity against these cells with near 100% cell viability at concentrations under investigation (0.1 ~ 20 μM), which rendered CSmicelles an efficient and safe carrier system (Figure 2C, Supporting Figure 8C). The combined index (CI) of DOX and RA on activated HSCs was calculated, and CI20 (20% inhibition ratio) of 0.47 demonstrated a synergistic effect of DOX and RA combination. To explore the internalization pathways of CSmicelles in activated HSCs, we performed the cell uptake study with DOX+RA-CSmicelles at 4 °C or in the presence of various endocytosis inhibitors. Uptake efficiency was markedly decreased at 4 °C (DOX: 76.2%, RA: 74.3%) or in the presence of sodium azide (DOX: 80.1%, RA: 78.6%) (Supporting Figure 9), suggesting an energy-dependent pathway. All inhibitors demonstrated different degree of suppression on the 7


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internalization of DOX+RA-CSmicelles in activated HSCs. Pre-incubation with CD44 antibody triggered the most significant uptake reduction (DOX: 66.1%, RA: 68.2%), followed by chlorpromazine (DOX: 29.7%, RA: 28.2%), filipin (DOX: 26.9%, RA: 25.1%), nystatin (DOX: 25.1%, RA: 23.7%), MβCD (DOX: 21.7%, RA: 22.3%), indomethacin (DOX: 17.5%, RA:15.8% ), and amiloride (DOX: 8.6%, RA: 8.1%). The functions of each inhibitor used herein were described previously42. Based on our results, the internalization of DOX+RACSmicelles was probably mediated by clathrin, and lipid raft/caveolae in addition to CD44 receptor dependent pathways. The high affinity between CSmicelles and CD44 was also confirmed by Figure 3A in which HSCs showed overlapped color (yellow) between CD44 and DOX (from CSmicelles), or between CD44 and FITC-labeled CSmicelles on the cell surface, but no overlaps for CD44 and DOX+RA-solution.

CSmicelles target Golgi mediated by N-acetylgalactosaminyl-transferase (GalNAc-T). Understanding the cellular distribution of nanomicelles is essential in achieving selective delivery of cargos to the subcellular organelles. After applying DOX+RA-solution, the fluorescence of organelle selective dyes (red) was distinctly separated from the fluorescence of DOX (green), indicating that DOX did not localize in lysosomes, mitochondria, endoplasmic reticulum or Golgi apparatus (Supporting Figure 10). However, DOX from DOX+RACSmicelles or FITC-labeled CSmicelles was found to localize in Golgi apparatus, suggesting that the CSmicelles were transported to Golgi apparatus after being endocytosed (Figure 3A and Supporting Figure 11). The fluorescence signals of lysosomes tracker, mitochondria tracker or endoplasmic reticulum tracker were distinctly separated from the fluorescence of DOX or FITC-labeled CSmicelles (green), indicating that CSmicelles were not localized in any of these organelles. Golgi apparatus is home to a multitude of glycosyltransferases (GTs), glycosidases, and nucleotide sugar that function together to complete the synthesis of glycans from attaching sugars covalently to proteins.43, 44 GalNAc-Ts specifically recognize and transfer GalNAc to the polypeptide chain.45, 46 GalNAc was the main component of CS,47 and we hypothesized that 8


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Golgi affinity of CSmicelles might be related to the specific binding of the transferase to CS. To test our hypothesis, we preincubated activated HSCs with 100 μg/mL GalNAc to saturate GalNAc-Ts for 30 min before DOX+RA-CSmicelle incubation. According to Figure 3B, the green fluorescence of DOX no longer co-localized with the red fluorescence of Golgi apparatus after GalNAc preincubation (+GalNAc), while the yellow fluorescence was observed in the cells without preincubation of GalNAc (-GalNAc ). Figure 4A also confirmed the binding of DOX+RA-CSmicelles and GalNAc-T. From quantitative analysis (Supporting Figure 12), the uptake of DOX from DOX+RA-CSmicelles decreased from 1087.13 ng/mL to 886.68 ng/mg after preincubation of GalNAc in HSCs. The uptake of RA from DOX+RA-CSmicelles reduced to 320.52 ng/mL in the presence of GalNAc while the untreated concentration was 396.21 ng/mL. Further, we used the GalNAc-T antibody to suppress GalNAc-T, 45% decrease in DOX uptake and 62% reduction in RA uptake with CSmicelles were observed, and no significant changes were found in DOX+RA-solution group. The same trend was observed in HepG2 cells (Supporting Figure 12). These results suggested GalNAc-T play an important role in CSmicelles mediated Golgi targeting. The inhibition, however, cannot reach 100%, mainly due to the inherent nucleophilicity of DOX. Nevertheless, our results supported that RA might destroy the Golgi structure and inhibit collagen synthesis in activated HSCs, while low-dose DOX could induce cell death in activated HSCs by disrupting DNA function. To further confirm Golgi targeting, Golgi apparatus was isolated using a commercially available kit and the drug concentration in Golgi was determined by LC-MS. In Figure 4B, GalNAc-T antibody pre-incubation led to a reduction in drug concentration in Golgi by approximately 75% of control at the same level of DOX+RA-micelles and DOX+RA-solution. Together, these results demonstrated CSmicelles had a high affinity with Golgi apparatus probably mediated by GalNAc-T.

RA destroys Golgi structure and reduces collagen secretion in HSCs. Interestingly, RA treated cells displayed different morphology of the Golgi apparatus as compared to that of control cells. Staining control cells with GM130 antibodies labeled the cis-Golgi, which showed 9


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that cis-Golgi localized in close proximity to cell nuclei (Figure. 4C). However, the GM130 labeled structures in RA treated (including RA-CSmicelles and DOX+RA-CSmicelles) cells did not localize in the perinuclear regions and became fragmented throughout the cell plasma (Figure. 4C). Thus, the polarity of the Golgi apparatus might be disentangled suggesting a potential fragmentation into mini-stacks. To determine whether RA-contained formulation impacted cisternal stacking, we quantitatively determined the cisternal numbers, which showed a significantly higher number (P < 0.05) in RA-CSmicelles or DOX+RA-CSmicelles treated activated HSCs than they were in the control group (Figure 4D). Transmission electron microscopy (TEM) further confirmed that the Golgi apparatus was divided into small stacks containing a similar number of cisternae in RA treated cells compared with the control cells (Figure. 4E). TEM images also displayed the appearance of relatively large vacuoles associated with the Golgi apparatus in RA treated cells. Next, cisternal numbers and lengths per stack was quantified. The result agreed with confocal micrograph that cisternal number per stack and length decreased significantly (P < 0.05) (Figure 4F and G). Fluorescence recovery after photobleaching (FRAP) assays was performed using a Golgi tracker, which showed that recovery was significantly inhibited in RA-CSmicelles or DOX+RA-CSmicelles treated activated HSCs as compared to control or DOX-CSmicelles group (Figure 4H and I). Together, these results indicated that RA treatment appeared to interrupt the structure of Golgi apparatus, thus rendering the Golgi apparatus into mini-stacks. RA is a Golgi-disturbing agent that can transform the Golgi apparatus into a diffuse vacuolar aggregate, and some components of the protein kinase C (PKC) play a vital role in the RAinduced Golgi apparatus disruption.48, 49 RA has high affinity to diacylglycerol, which binds to the diacylglycerol-sensitive allosteric enzyme center of PKC.50 PKC regulates the function of many membranous organelles and cytoskeletal components.51 The dysfunction of PKC results in an imbalance in vesicle fusion and budding at the Golgi apparatus.52 Collagen is the most abundant protein in mammals and is a main component of extracellular matrix (ECM) which provides a vital structural matrix for tissue maintenance, development, and regeneration.53 Normal tissue development involves dynamic collagen remodeling 10


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processes including both collagen production and degradation.54 Imbalance in these processes causes structural and metabolic abnormalities of collagen, and leads to various pathological conditions, like fibrosis, arthritis, and cancer.55 Thus, detecting anomalies in collagen would be significant in diagnosing and treating related pathological conditions. The Sirius Red (SR) staining procedure selectively stains Type I collagen fibers, as well as Type III reticular fibers.56 A comparison of the secretion of collagen in different preparations was done and it was found that the amount of activated HSCs stained with red light was the least in the group treated with DOX+RA-CSmicelles or RA-CSmicelles (Figure 5A). After immunofluorescent staining of type I collagen, the red fluorescence of cells from groups treated with DOX+RA-CSmicelles or RA-CSmicelles was also weaker than DOX+RA-solution, DOX+RA-micelles, and DOXCSmicelles (Figure 5B). Then we compared the amount of collagen in the culture medium by SR staining, Figure 5C and D showed that collagen amount of DOX+RA-CSmicelles or RACSmicelles group was significantly lower than control. Additionally, RA-included CSmicelles also downgraded the expression of type I collagen (Figure 5E and F), suggesting that RACSmicelles functionally targeted Golgi apparatus, destroyed the Golgi structure and reduced the collagen secretion and expression of HSCs.

CSmicelles selectively accumulate in the liver in fibrotic rats. We first isolated various cell populations from either normal or injured livers in rats and examined CD44 expression levels using flow cytometry. As shown in Supporting Figure 13, normal liver cells barely expressed CD44, whereas the fibrotic liver showed 8 times higher levels of CD44 than in the normal liver. We then carried out in vivo imaging of healthy and fibrotic model rats following the administration of CSmicelles. Figure 6 A and Supporting Figure 14 showed the fluorescent images of dissected organs from healthy or fibrotic rats after tail-vein injections. Whether in healthy or fibrotic rats, DOX+RA-CSmicelles accumulated mainly in the liver. Compared to the healthy rats, however, the clearance of DOX+RA-CSmicelles was relatively slow in a cirrhotic liver, mainly due to the following reasons: i) HSCs activated and proliferated in the fibrotic liver thus increased the binding sites of CS; ii) the fibrotic liver became dysfunctional 11


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and unable to clear DOX+RA-CSmicelles efficiently. When micelles were modified with CS, they were endowed with HSCs selectivity. HSCs activated and proliferated in the fibrotic liver thus resulting in the increased binding sites of CS. By comparing different formulations in fibrotic rats, we found that the fluorescent intensity of DOX+RA-CSmicelles treated liver was much stronger than DOX+RA-micelles and DOX+RA-solution (Figure 6A, Supporting Figure 14). The fluorescence of DOX+RA-solution was negligible, reflecting the clearance through reticuloendothelial systems of liver, spleen, and kidney in 1 h. When the rats were pretreated with CS solution, accumulation of DOX+RA-CSmicelles in liver decreased obviously. However, in DOX+RA-micelles group, pretreatment of CS solution resulted in no changes, which suggested that CS rendered the micellar system with active target ability to the liver. To gain insights into the biodistributions of DOX and RA, we used LC-MS to determine drug concentrations in biological samples. At each given time point, DOX+RA-CSmicelles group showed significantly higher DOX concentration than that of DOX+RA-micelles and DOX+RA-solution in plasma (P < 0.05) (Supporting Figure 15A and B). The pharmacokinetic parameters of DOX and RA were summarized in Supporting Table 3. For the pharmacokinetic profiles of DOX, compared with DOX+RA-solution (Cmax, 935.1 ± 71.4; AUC0–∞, 40216.2 ± 2123.4 ng·min/mL), DOX+RA-CSmicelles group showed significantly higher Cmax (3313.3 ± 201.9 ng/mL), and AUC0–∞ (119947.5 ± 69212.3 ng·min/mL) (P < 0.05). Subsequently, the distribution profiles of DOX+RA-CSmicelles in rats at 0.25, 1, and 2 h following intravenous injection were shown in Supporting Figure 15C and 15D. The intravenously administered DOX+RA-CSmicelles mainly distributed in liver, lung, and kidney in rats. Especially in liver, the DOX+RA-CSmicelles group showed that the DOX concentration was significantly higher than DOX+RA-solution by 1.30-, 1.34-, and 1.91-fold at 0.25, 1, and 2 h respectively, which may benefit the treatment of liver diseases. In contrast, DOX+RAmicelles group showed a significantly higher level of DOX distribution in kidney than for other groups after administration (P < 0.05), indicating that unmodified normal micelles were easily removed by kidneys.

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To combat fibrosis, the ultimate target of DOX+RA-CSmicelles in liver is activated HSCs. The amount of CD44 expression on different liver cells were further elucidated by laser confocal microscopy. Activated HSCs, hepatic parenchymatous cells (HPCs), Kupffer cells, and hepatic sinusoidal endothelial cell (HSECs) were stained with α-SMA, HNF-1β, F4/80, and CD31 antibodies, separately. In Supporting Figure 16, HSCs and Kupffer cells proliferated more in the fibrotic liver leading to upregulated levels of CD44. Almost all CD44 was colocalized with HSCs and partly overlapped with Kupffer cells. Then, we determined the cellular localization of the DOX+RA-CSmicelles in the fibrotic (Figure 6B) or healthy rats’ liver (Figure 6C). In the confocal images, DOX was shown in green and the cell markers were stained in red. The cell nuclei were counter-stained with DAPI. In the DOX+RA-CSmicelles group, DOX was found to merge with HSCs to generate strong yellow signals, indicating that most DOX molecules were located in HSCs. DOX was partly co-localized with Kupffer cells (stained by F4/80), which was related to the CD44 expression on Kupffer cells. Kupffer cells are nonparenchymal cells in the liver with a variety of biological functions playing a crucial role in liver fibrosis.57 Kupffer cells secrete pro-fibrotic factors such as transforming growth factor β1 (TGF-β1) and platelet-derived growth factor (PDGF) to activate HSCs into myofibroblasts, which then synthesize collagen and metalloproteinase inhibiting factor 1 (TIMP-1) during fibrosis.58,59 Thus, the incorporation of Kupffer cells and DOX+RA-CSmicelles may benefit the curing of liver fibrosis. By comparison, the fluorescence of DOX+RA-solution and DOX+RA-CSmicelles in liver was relatively weak and irregular. Taken together, these results clearly demonstrated that, when administrated via the circulation, DOX+RA-CSmicelles could target, and deliver drugs to activated HSCs in vivo.

DOX+RA-CSmicelles demonstrate antifibrosis activity in rats. The ultimate purpose of targeted delivery of DOX and RA to activated HSCs was to treat liver fibrosis. After entering HSCs, the drugs were expected to be released from the micelles and suppress the expression of pathological genes and prevent the development of liver fibrosis. We set up a CCl4-induced liver fibrosis in rats to evaluate the therapeutic performance of DOX+RA-CSmicelles, with 13


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DOX+RA-micelles, DOX+RA-solution, DOX-CSmicelles, RA-CSmicelles, and saline as comparisons. The entire course of treatment was illustrated in Supporting Table 4. We performed a stringent therapeutic test of DOX+RA-CSmicelles on CCl4 fibrosis model rats.6 The fibrosis animal models received the treatment 4 weeks after the models were given CCl4 for liver fibrosis induction. Preparations were given to the animals twice a week for 4 weeks. CCl4 was administrated until the end of the experiment. Meanwhile, the rat body weight was recorded, the alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), total bile acid (TBA), albumin (ALB), triglyceride (TG), total cholesterol (TC), and glutathione peroxidase (GSP-PX) levels in the sera were examined. Investigating the effect of CCl4 on serum biochemical parameters, significant (P < 0.05) enhancement in the levels of AST, and ALT were observed in the sera of CCl4-injected rats. At the end of the therapy (at the 8th week), treatment with DOX+RA-CSmicelles (0.5 mg/kg) almost reinstated CCl4-mediated abnormalities of serum biochemical parameters (TBIL, TBA, ALB, TG, TC, and GSP-PX) near to normal levels (P > 0.05) (Supporting Figure 17). Regarding liver morphology (Figure 7A), obscure boundary and scleroid changes were observed in the saline group with a pink color. The liver treated with DOX+RA-CSmicelles was in crimson color and appeared resembling to healthy liver. Histological analysis of the rat liver tissue sections suggested that DOX+RA-CSmicelles significantly reduced the number of highly proliferative HSCs, collagen fibers deposition (Figure 7B, C, D, E and F), and inflammatory lesions (Supporting Figure 18). From quantitative analysis of type I collagen (Figure 7G and H) and hydroxyproline (Figure 7I) in liver tissue, DOX+RA-CSmicelles group showed significantly lower amount than DOX-CSmicelles, RA-CSmicelles, DOX+RA-micelles, DOX+RA-solution and saline group (P < 0.05). In above tests, the RA significantly improved the therapeutic performance of DOX, in comparison with the naked DOX group. Treatment with DOX+RACSmicelles achieved successful inhibition of the development of hepatic fibrosis. Supporting Figure 19 showed the efficacy of various formulations on fibrosis mice at 6th (A) and 7th (B) week. The data indicated that low-dose DOX+RA-CSmicelles could rival the damage of CCl4 and had a preferred protective effect on the liver during the experimental period. 14


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DOX, as an anticancer drug in clinical application, is limited by side effects such as cardiac toxicity and myelosuppression.60-62 According to the safety evaluation data, animals treated with all formulations exhibited no significant weight loss (Supporting Figure 20), bone cardiac toxicity and marrow suppression (Supporting Figure 21). Low-dose DOX administered via a targeted delivery system may contribute to the therapeutic efficacy in vivo. Targeted delivery of antitumor drugs provided higher accumulations of the antitumor drug in the liver with reduced systemic drug exposure, and the incidence of adverse effects may be decreased. Together, the combination of DOX and RA were proven potent inhibitors of HSCs activation and proliferation in vivo, inhibiting cellular proliferation in the nanomolar concentration range. DOX was administered at a relatively low dose than many other typical inhibitors of HSCs proliferation, such as statins, the selective Na/H+ exchange inhibitor cariporide, mycophenolic acid, and the semisynthetic analog of fumagillin TNP-470.63-65 The outstanding antifibrosis effect of DOX and RA combination may relate to multiple intracellular mechanisms including Golgi apparatus disaggregation and nucleic acid inhibition.

CONCLUSIONS In summary, CS derivatives were synthesized and applied to fabricate CS based nanomicelles as HSCs-specific carriers for the treatment of liver fibrosis. DOX and RA loaded CSmicelles were assembled using thin-film hydration method, which showed selective internalization in activated HSCs and hepatoma cells other than in normal liver cells. Also, CSmicelles accumulated in Golgi apparatus and delivered cargos specifically to Golgi mediated via GalNAc-T. RA was then proven to destroy the Golgi structure and inhibit collagen synthesis in activated HSCs, while low-dose DOX can induce cell death in activated HSCs by disrupting DNA function. Furthermore, DOX+RA-CSmicelles were delivered in a target specific manner and accumulated more efficiently in the fibrotic liver than in the normal liver following systemic administration. Together, CSmicelles have been well demonstrated as a Golgi-specific carrier that can selectively deliver therapeutics to HSCs mediated via CD44 receptors, which represents a highly promising delivery system for the targeted therapy of chronic liver diseases. 15


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MATERIALS AND MATHODS Materials. CS (Mw 120,000 Da), deoxycholic acid (DOCA), 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyl tetrazolium bromide (MTT), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), DOX·HCl, and RA were purchased from Sigma-Aldrich (USA). Phospholipid (E80) was purchased from Lipoid (Germany). Cell culture medium was bought from GIBCO (USA). HSCs, HepG2, HUVEC, and LO2 cell lines purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China).

Chondroitin Micelle Preparation. Blank CSmicelles, DOX-CSmicelles, RA-CSmicellles, DOX+RA-CSmicelles and DOX+RA-micelles, were assembled using the thin-film hydration method as previously described.66 Briefly, each material was dissolved in 10 mL methanol in appropriate proportion within 25 mL clear glass flask. After desiccation to remove the solvent, the resulting thin films were hydrated in 1 mL of phosphate buffer saline (PBS).

Characterization. The particle size, PDI and ξ-potential values of micelles were measured by dynamic light scattering (DLS) using Malvern Zetasizer (Malvern, NanoZS90, UK). The size and morphology of micelles were examined using Tecnai G2 F20 S-TWIN transmission electron microscopy (FEI, USA). The micelles were stained with 1% uranyl acetate. The analysis of DOX and RA was performed with high performance liquid chromatography (HPLC) system with a UV detector (Agilent Technologies Inc, Cotati, CA). To estimate the encapsulation efficiency of micelles, the micelle structure was destroyed by adding methanol. The solution was properly diluted prior to HPLC analysis. Drug-loading coefficient (DL) and encapsulation efficiency (EE) and were calculated with the following formulas: DL% = (weight of drug measured in micelles/(total weight of micelle materials added + weight of the feeding drug)) × 100%, EE% = (weight of drug measured in micelles/weight of the feeding drug) × 100%.

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Subcellular localization. To determine the subcellular distribution, we performed triplelabeling experiments using confocal microscopy in activated HSCs. The localization of DOX or DOX+RA-CSmicelle in subcellular organelles was visualized by labeling the HSCs with fluorescent probes specific to the organelle marker such as Mito Tracker, Lyso Tracker, ER tracker and Golgi tarker (Sigma, USA). Cells were seeded into chambered coverslips and cultured for 24 h at 37 °C in the presence of 5% CO2, followed adding 5 μM of free DOX and RA, DOX+RA-CSmicelles or unencapsulated CSmicelles (FITC labeled), respectively. The drug containing medium was removed after incubated for 2 h, and cells were washed with cold PBS for three times. Then the cells were stained with organelle-selective probes. Golgi apparatus, mitochondria, endoplasmic reticulum (ER) and lysosomes were visualized by staining the cells with 10 mM BODIPY TR ceramide complexed to BSA, 100 nM Mito Tracker Deep Red FM, 2 mM ER Tracker Red, and 100 nM Lyso Tracker Red DND-99 for 30 min, respectively. The cell nuclei were stained with DAPI for 10 min. Then the cells were washed three times with PBS and observed with confocal microscope. To explore whether the Golgi apparatus-targeting is driven by GalNAc-T, we saturated the GalNAc-T by adding 100 μM GalNAc to activated HSCs 30 min before DOX+RA-CSmicelles incubation. The cells without GalNAc pre-incubated were set as control. Then the cells were stained by protocols as mentioned above and observed with confocal microscope.

Electron microscopy. Activated HSCs were grown to ∼80% confluence on a 55 cm2 culture dish. After washing with PBS, cells were incubated with different formulations (1 μM) for 24 h at 37 °C. Cells were then washed with PBS for three times, fixed with 2% glutaraldehyde for 60 min and postfixed with 1% OsO4 at room temperature, followed by 1% uranyl acetate. The samples were dehydrated by a series of graded ethanol concentrations and embedded in Epon. The sections were contrasted with lead citrate, and viewed on TEM.

Cytotoxicity. To compare the cytotoxicity of DOX+RA-solution and various micelles in this study, MTT assay was performed. Firstly, cells in logarithmic growth phase were seeded in 9617


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well culture plates at approximate 6000 cells per well. After 24 h, cells were exposed to 200 μL of culture medium containing different concentrations (0.20, 0.5, 1.25, 2.5, 5, 10, 20 µM DOX) of DOX+RA-solution, various drug loaded micelles and blank micelles for another 48 h. Cells treated with blank culture medium served as negative control group and the well without cell served as blank group. Finally, the cell viability was determined by standard MTT assay as previously described.67

Liver Fibrosis Rat Model. Eight-week-old male Wistar rats were purchased from Dashuo Biotechnology (China) and reared in specific pathogen-free conditions. All animal procedures were approved by the ethics committee of Sichuan University and conducted in accordance with university guidelines. Animals were treated, twice weekly, with CCl4 at a dose of 100 mg/kg (prepared with olive oil 1:1 v/v). After four weeks, animals developed liver fibrosis as validated by pathological analysis.

Therapeutic efficiency. Fibrotic rats were assigned to seven groups at random, with ten rats in each. Rats without any treatment were as controls. The dosing regimen was showed in Supporting Table 4. During the 8 weeks of the study, the rats were observed and their body weight was measured twice weekly. Bone marrow suppression is one of the major adverse effects of chemotherapy. To assess the suppression level caused by DOX+RA-solution or DOX+RA-CSmicelles, the blood samples were collected on week 6, 7 and 8. RBC and WBC were counted by MEK-6318K Automated Hematology Analyzer (Nihonkohden, Shinjuku, Japan). Serum AST, ALT, TBIL, TBA, ALB, TG, TC, and GSH-PX levels were measured by an AU480 Chemistry Analyzer (Beckman Coulter, USA) according to IFCC primary reference procedures. The rats were sacrificed 72 h after the administration of the last dose of preparation. The liver and heart were removed and fragments of these organs were fixed in 4% paraformaldehyde, sectioned and stained for histopathology and immunohistochemistry analysis.

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Biodistribution. Liver fibrosis models were established as described above. Rats were randomly divided into five groups: DOX+RA-solution, DOX+RA-micelles, DOX+RACSmicelles, DOX+RA-micelles (pretreated with 10 mg/kg CS solution) and DOX+RACSmicelles (pretreated with 10 mg/kg CS solution) at an equivalent dose of 2 mg/kg DOX, and saline as control. The rats were sacrificed at 0.25 h, 1 and 2 h post injection (n = 5 per each group), major organs including heart, liver, lung, spleen and kidney were collected, washed, and weighed. The tissue fluorescent images were obtained through Caliper IVIS Lumina III (Perkin elmer, USA). The scanning parameters were as follows: excitation wavelength = 500 nm, emission wavelength = 600 nm, fluency rate = 2 mW/cm2, and field of view = 13.5 cm. The camera was set to maximum gain, a luminescent exposure time of 4 s, and a binning factor of 4. Then, for further quantitative analysis, weighted tissues were homogenized with 3-fold of acetonitrile on the Precellys 24 lysis instrument. The tissue homogenates were centrifuged at 12,000 rpm for 10 min. The drug concentration in the supernatant was determined by LC-MS, and the biodistribution of DOX and RA in each organ was normalized by the weight of the selected tissues.

Masson and hematoxylin-eosin (H&E) assay. To assess the therapeutic effects of various therapy formulation, the pathological state in rats with liver fibrosis following treatment was detected by the Masson method and H&E staining. The slides were observed by confocal microscope.

Immunofluorescence and Immunohistochemistry. Cells were seeded on glass coverslips 24 h before the experiments. Following treatment, the cells were washed with PBS for 10 min and fixed in 4% paraformaldehyde for 20 min. Cells were then washed and permeabilized with 0.1% (v/v) Triton X-100 for 5 min. PBS with 5% (v/v) bovine serum albumin (BSA) was used as blocking solution for 1 h, followed incubating samples with primary antibodies in blocking solution overnight at 4 °C. Cells were then washed with blocking solution and incubated with fluorophore-labeled secondary antibodies for 1 h at room temperature. After a final washing 19


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step, the samples were incubated in DAPI for 10 min. Images of the specimens were acquired using confocal microscope and quantified using the ImageJ software. The CD44, α-SMA, CD31, HNF-1, F4/80 expression in the activated HSCs of liver fibrotic rats was evaluated using corresponding antibody (Abcam, USA) at a 200-fold dilution and detected by immunofluorescence. After treatment with the different formulations, livers were harvested and prepared for paraffin-embedded sectioning. Tissue sections were counterstained with DAPI for nuclei coloration and observed by confocal microscopy.

Western blot. For determination of protein abundance by Western blot, activated HSCs or liver tissue were placed in lysis buffer supplemented with protease inhibitor (1 mg/ml), 0.5% sodium orthovanadate, 10% SDS, and 0.5% sodium fluoride. Lysates were loaded on 6% SDSpolyacrylamide gel (ThermoScientific, USA), blotted onto polyvinylidene fluoride membrane (0.45 μm, Millipore, USA), and blocked with 5% BSA in tris-buffered saline with Tween-20 (0.1%) for 30 min. Blots were then incubated with primary antibodies recognizing type I Collagen (Sigma-Aldrich, USA) overnight at 4 °C, washed, incubated with appropriate secondary horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature; and developed with Chemiluminescent substrate (ThermoScientific, USA). Blots performed in quintuplicate were imaged and quantified using ImageJ software with densitometry analysis.

Statistical Analysis. A minimum of two independent experiments were studied, with 3 - 12 rats per treatment group in each experiment (n = 3 - 12). ANOVA and two-tail Student t tests were performed to determine statistical significance.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Tao Gong: 0000-0002-9866-9911 20


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Conflict of Interest: The authors declare no competing financial interest.

Acknowledgment: This project is financially supported by grants from the National Natural Science Foundation of China (No. 81690261 and 81673359).

Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthesis and characterization of polymers, LC-MS analysis method, in vitro assays of the micelles, SR staining, hydroxyproline determination, cellular uptake, supporting tables 1-4 and supporting figures 1-22 (PDF)

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Sanchez-Ramos, J.; Patel, N. A. Vitamin A Metabolite, All-Trans-Retinoic Acid, Mediates Alternative Splicing of Protein Kinase C DeltaVIII (PKCdeltaVIII) Isoform Via Splicing Factor SC35. J. Biol. Chem. 2010, 285, 25987-2595. 53.

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Vitro and in Vivo. Mol. Pharmaceutics 2015, 12, 274-286.

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Figure 1. Schematic representation for the assembly of DOX+RA-CSmicelles targeting activated HSCs by CD44 mediated endocytosis and then targeting Golgi apparatus by GalNAcT mediation.

Figure 2. (A) Confocal microscopic images of activated HSCs and LO2 cells incubated for 2 h at 37 °C with DOX+RA-CSmicelles, DOX+RA-micelles, and DOX+RA-solution. Bar, 50 μm. (B) Quantitation of DOX and RA uptake by activated HSCs and LO2 cells (n = 3, mean ± SD). *P < 0.05 vs DOX+RA-micelles, $P < 0.05 vs DOX+RA-solution. (C) Inhibitory effects of six formulations on the proliferation of activated HSCs and LO2 cells measured by MTT assay (n = 3, mean ± SD).

Figure 3. (A) Distribution of DOX from DOX+RA-CSmicelles in several cytoplasmic organelles of activated HSCs. (B) Distribution of DOX from DOX+RA-CSmicelles in Golgi apparatus of activated HSCs pre-incubated with or without GalNAc. Bar, 1 μm.

Figure 4. (A) Fluorescent images of GalNAc-T (red fluorescence) stained HSCs treated with DOX+RA-CSmicelles, DOX+RA-micelles, or DOX+RA-solution (green fluorescence), respectively. Bar, 5 μm. (B) Drug concentration changes in isolated Golgi apparatus preincubated with GalNAc or GalNAc-T antibody. *P < 0.05 vs control. (C) Confocal microscopic images of cis-Golgi apparatus marked by GM130 in activated HSCs treated with DOX-CSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. Bar, 5 μm. (D) Scatter plots show the number of Golgi fragments in activated HSCs after treated with DOX-CSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. Each dot represents values from a single cell. **P < 0.01. (E) Transmission electron microscopic images of Golgi apparatus treated with DOXCSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. The arrowheads indicate Golgi structures. Bar, 1μm. Bar graphs below show the average number of cisternae per stack (F) and cisternal lengths (G). *P < 0.05 vs control. n > 10 cells per group. (H) Pseudocolored images of FRAP assays using the Golgi tracker in activated HSCs treated with DOX-CSmicelles, RA-

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CSmicelles, or DOX+RA-CSmicelles at time points. Bar: 1 μm. (I) The line chart shows the intensity recovery profile (%) after photobleaching (n = 20 cells, mean ± SD).

Figure 5. (A) Sirius Red staining of activated HSCs treated with PBS, DOX+RA-solution, DOX+RA-micelles, DOX-CSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. Bar, 50μm. (B) Immunofluorescent staining of type I collagen in activated HSCs treated with above formulations. Bar, 25 μm. Quantitative analysis of type I and III collagen in serum-free medium of activated HSCs treated with above formulations at the dose of 0.1 μg/ml (C) and 1μg/ml (D) (n = 5, mean ± SD). *P < 0.05. (E) Western blot analysis of type I collagen levels in activated HSCs. β-Actin was included as loading control. (F) Quantitative analysis of type I collagen in activated HSCs (n = 5, mean ± SD). *P < 0.05 vs DOX+RA-solution, #P < 0.05 vs DOX+RAmicelles, $P < 0.05 vs DOX-CSmicelles.

Figure 6. (A) Ex vivo DOX fluorescence images showing the bio-distribution of DOX+RAsolution, DOX+RA-micelles and DOX+RA-CSmicelles in healthy rats or liver fibrotic rats at 0.25 h after injection. The liver tissue from liver fibrotic rats (B) or healthy rats (C) was then sectioned and immunofluorescent stained by α-SMA (for HSCs), CD31 (for HSECs), F4/80 (for Kupffer cells), or HNF-1β antibody (for HPCs), and DAPI for cell nucleus (blue). The fluorescent DOX was shown in green. Co-localization was determined where the green and red fluorescence perfectly merged into yellow signals. The fluorescent photographs were taken by confocal microscopy. Bar, 100 μm.

Figure 7. (A) Representative photos of liver appearance from healthy rats or liver fibrotic rats receiving various treatments. (B) Panoramic scanning, (C) Masson staining, (D) H&E staining, (E) α-SMA immunofluorescent staining, and (F) IL-1β immunohistochemistry of liver from rats following different treatments. Bar, 100μm. (G) Collagen area of liver stained by Masson and calculated by ImageJ software. (n = 15, mean ± SD). (H) Western blot analysis of collagen I levels in liver tissues. β-actin was included as loading control. (I) Quantitative analysis of type I collagen in liver tissues (n = 5, mean ± SD). *P < 0.05 vs DOX+RA-solution, #P < 0.05 vs 28


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DOX+RA-micelles, $P < 0.05 vs DOX-CSmicelles, $P < 0.05 vs RA-CSmicelles. (J) Hydroxyproline amount in liver tissues with different treatments (n = 10, mean ± SD).

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Figure 1. Schematic representation for the assembly of DOX+RA-CSmicelles targeting activated HSCs by CD44 mediated endocytosis and then targeting Golgi apparatus by GalNAc-T mediation.


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Figure 2. (A) Confocal microscopic images of activated HSCs and LO2 cells incubated for 2 h at 37 °C with DOX+RA-CSmicelles, DOX+RA-micelles, and DOX+RA-solution. Bar, 50 μm. (B) Quantitation of DOX and RA uptake by activated HSCs and LO2 cells (n = 3, mean ± SD). *P < 0.05 vs DOX+RA-micelles, $P < 0.05 vs DOX+RA-solution. (C) Inhibitory effects of six formulations on the proliferation of activated HSCs and LO2 cells measured by MTT assay (n = 3, mean ± SD).


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Figure 3. (A) Distribution of DOX from DOX+RA-CSmicelles in several cytoplasmic organelles of activated HSCs. (B) Distribution of DOX from DOX+RA-CSmicelles in Golgi apparatus of activated HSCs pre-incubated with or without GalNAc. Bar, 1 μm.


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Figure 4. (A) Fluorescence images of GalNAc-T (red fluorescence) stained HSCs treated with DOX+RACSmicelles, DOX+RA-micelles, or DOX+RA-solution (green fluorescence), respectively. Bar, 5 μm. (B) Drug concentration changes in isolated Golgi apparatus preincubated with GalNAc or GalNAc-T antibody. *P < 0.05 vs control. (C) Confocal microscopic images of cis-Golgi apparatus marked by GM130 in activated HSCs treated with DOX-CSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. Bar, 5 μm. (D) Scatter plots show the number of Golgi fragments in activated HSCs after treated with DOX-CSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. Each dot represents values from a single cell. **P < 0.01. (E) Transmission electron microscopic images of Golgi apparatus treated with DOX-CSmicelles, RA-CSmicelles and DOX+RACSmicelles. The arrowheads indicate Golgi structures. Bar, 1μm. Bar graphs below show the average number of cisternae per stack (F) and cisternal lengths (G). *P < 0.05 vs control. n > 10 cells per group. (H) Pseudocolored images of FRAP assays using the Golgi tracker in activated HSCs treated with DOXCSmicelles, RA-CSmicelles, or DOX+RA-CSmicelles at time points. Bar, 1 μm. (I) The line chart shows the intensity recovery profile (%) after photobleaching (n = 20 cells, mean ± SD).


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. (A) Sirius Red staining of activated HSCs treated with PBS, DOX+RA-solution, DOX+RA-micelles, DOX-CSmicelles, RA-CSmicelles and DOX+RA-CSmicelles. Bar, 50 μm. (B) Immunofluorescent staining of type I collagen in activated HSCs treated with above formulations. Bar, 25 μm. Quantitative analysis of type I and III collagen in serum-free medium of activated HSCs treated with above formulations at the dose of 0.1 μg/ml (C) and 1 μg/ml (D) (n = 5, mean ± SD). *P < 0.05. (E) Western blot analysis of type I collagen levels in activated HSCs. β-Actin was included as loading control. (F) Quantitative analysis of type I collagen in activated HSCs (n = 5, mean ± SD). *P < 0.05 vs DOX+RA-solution, #P < 0.05 vs DOX+RA-micelles, $P < 0.05 vs DOX-CSmicelles.


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Figure 6. (A) Ex vivo DOX fluorescence images showing the bio-distribution of DOX+RA-solution, DOX+RAmicelles and DOX+RA-CSmicelles in healthy rats or liver fibrotic rats at 0.25 h after injection. The liver tissue from liver fibrotic rats (B) or healthy rats (C) was then sectioned and immunofluorescent stained by α-SMA (for HSCs), CD31 (for HSECs), F4/80 (for Kupffer cells), or HNF-1β antibody (for HPCs), and DAPI for cell nuclei (blue). The fluorescent DOX was shown in green. Co-localization was determined where the green and red fluorescence perfectly merged into yellow signals. The fluorescent photographs were taken by confocal microscopy. Bar, 100 μm.


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Figure 7. (A) Representative photos of liver appearance from healthy rats or liver fibrotic rats receiving various treatments. (B) Panoramic scanning, (C) Masson staining, (D) Hematoxylin-eosin staining, (E) αSMA immunofluorescent staining, and (F) IL-1β immunohistochemistry of liver from rats following different treatments. Bar, 100 μm. (G) Collagen area of liver stained by Masson and calculated by ImageJ software. (n = 15, mean ± SD). (H) Western blot analysis of type I collagen levels in liver tissues. β-actin was included as loading control. (I) Quantitative analysis of type I collagen in liver tissues (n = 5, mean ± SD). *P < 0.05 vs DOX+RA-solution, #P < 0.05 vs DOX+RA-micelles, $P < 0.05 vs DOX-CSmicelles, &P < 0.05 vs RA-CSmicelles. (J) Hydroxyproline amount in liver tissues with different treatments (n = 10, mean ± SD).


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ToC graphic


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