SB431542 | Survivin Signal

SB431542-Loaded Liposomes Alleviate Liver Fibrosis by Suppressing TGF‑β Signaling
Jinhang Zhang, Rui Li, Qinhui Liu, Jian Zhou, Hui Huang, Ya Huang, Zijing Zhang, Tong Wu, Qin Tang, Cuiyuan Huang, Yingnan Zhao, Guorong Zhang, Li Mo, Yanping Li,* and Jinhan He*

ACCESS
Metrics & More
Article Recommendations
*sı Supporting Information

⦁ INTRODUCTION
Liver ﬁbrosis and cirrhosis are the major causes of morbidity and mortality in chronic liver disease.1,2 If unmanaged, liver ﬁbrosis may develop into decompensated cirrhosis, and various end- stage liver disease complications may occur, such as gastro- intestinal bleeding, infection, hepatic encephalopathy, and liver and kidney syndrome.3 Despite signiﬁcant advances in under- standing hepatic ﬁbrosis and deﬁning targets for therapy, few antiﬁbrotic drugs have been approved for clinical use.4−6 Thus, the development of eﬀective antiﬁbrotic drugs and appropriate drug delivery systems are urgently needed.5
Activation and proliferation of hepatic stellate cells (HSCs) are known as the central driver of liver ﬁbrogenesis.7,8 Upon liver injury in vivo or cell culture in vitro, HSCs are activated and become myoﬁbroblasts, which produce excessive extracellular matrix (ECM).9 Transforming growth factor-β (TGF-β) is a master regulator of HSC activation and an essential cytokine to induce the ﬁbrotic response.10,11 TGF-β can induce the phosphorylation of Smad2/3, which in turn promotes HSC activation and upregulates the expression of ﬁbrotic genes.12,13 Thus, blocking TGF-β synthesis or suppressing its downstream pathway is an eﬀective way against liver ﬁbrosis.14
SB431542 is a synthetic small molecule that speciﬁcally inhibits TGF-β signaling mediated by ALK4, ALK5, or ALK7.15 It binds to the ATP binding site of type I receptor kinases and blocks the phosphorylation of its downstream eﬀectors Smad2 and Smad3.16 Although SB431542 can selectively and eﬀectively
inhibit the activity of the TGF-β signaling pathway, several undesirable properties of SB431542, such as poor water solubility and low bioavailability, have greatly limited its therapeutic utility.
We speculated that loading SB431542 into liposomes may be a promising way to increase its water solubility and improve its pharmacokinetic properties. This approach might also allow targeting of the drug to ﬁbrotic tissues by the “enhanced permeability and retention” (EPR) eﬀect.17,18 Collagens in the space of Disse in ﬁbrotic liver distort the lumen and form ﬁbrotic septa, which restrict the microvascular blood ﬂow and result in hypoxia.19 This upregulates the vascular endothelial growth factor and receptors in HSCs, leading to angiogenesis of distorted vessels, which recruit nanoparticles through the EPR eﬀects similar to solid tumors.20
Using a simple thin-ﬁlm hydration method, we prepared liposomes with two excipients approved by the US Food and Drug Administration, soya phosphatidyl S100 and the poly- (ethylene glycol) (PEG)-based non-ionic surfactant Solutol HS15. Both excipients are proven eﬃcient to make poorly

Downloaded via RICE UNIV on October 26, 2020 at 02:40:50 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
© XXXX American Chemical Society
A

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

Mol. Pharmaceutics XXXX, XXX, XXX−XXX

soluble drugs suitable for intravenous administration.21 We show here that loading SB431542 into liposomes signiﬁcantly increased its solubility and improved pharmacokinetic proper- ties after an intravenous injection. The liposomes delivering SB431542 showed better eﬃcacy in alleviating liver ﬁbrosis either in vivo or in vitro via inhibition of TGF-β signaling.
⦁ MATERIALS AND METHODS
⦁ Materials and Animals. SB431542 (98% pure) was purchased from Selleck (Ontario, Canada); soya phosphatidyl (Lipoid S 100) was purchased from Lipoid (Ludwigshafen, Germany); Solutol HS 15 was purchased from BASF (Ludwigshafen, Germany); oleic acid (OA) was purchased
from Shinyo Pure Chemicals (Osaka, Japan); and 1,1′- dioctadecyl-3,3,3′,3′-tetramethylindodicarbo-cyanine (DiD) was purchased from Biotium (Hayward, USA). MTT was
purchased from Sigma-Aldrich (St. Louis, USA).
Mice and rat were housed at the West China Hospital, Sichuan University, in accordance with the guidelines of the Institutional Animal Care and Use Committee.
⦁ Preparation and Characterization of SB431542- Loaded Liposomes. SB431542-loaded liposomes (SB-Lips) were prepared by the thin-ﬁlm hydration method, as described.22 Brieﬂy, SB431542 (1 mg) and 60 μL 50 mg/mL OA were dissolved separately in 1 mL ethanol. After mixing the two solutions and stirring them at room temperature for 30 min, the solution of SB431542 and OA were transferred to a round- bottom ﬂask. Subsequently, the solvent was evaporated at 35 °C to form a thin layer of uniform ﬁlm. Then, S100 (25 mg) and Solutol HS 15 (6 mg), each dissolved in 3 mL ethanol, were added to the ﬂask. The organic phase was removed by vacuum rotary evaporation, and the ﬁlm was hydrated in 1 mL 5% glucose. The predispersion was intermittently sonicated using a probe sonicator at 250 W for 2 min to obtain SB-Lips.23 DiD- loaded liposomes (DiD-Lips) were prepared in the same way, except that SB431542 was replaced by DiD.
Size distribution and zeta potential of liposomes were
measured by dynamic light scattering with a Zetasizer Nano ZS90 instrument (Malvern, UK). The morphology of liposomes was examined by transmission electron microscopy (H-600, Hitachi, Japan) after staining with 2% phosphotungstic acid. To determine the encapsulation eﬃciency24 (EE %) and drug loading (DL %), free SB431542 was ﬁrst removed by ultraﬁltration, then the concentration of entrapped SB431542 was determined by high-performance liquid chromatography (HPLC) on a Waters 2996 system (Waters, USA), equipped with a reversed-phase C18 column (5 μm, 15 × 4.6 mm). The mobile phase was methanol/water (60/40, v/v), and the ﬂow rate was 1.0 mL/min. The drug was detected at 330 nm.
EE % was calculated using the following equation: EE % = (weight of drug in ﬁnal liposomes/weight of drug initially added to liposomes) × 100%. DL % was calculated using the following equation: DL % = (weight of drug in ﬁnal liposomes/total weight of ﬁnal liposomes) × 100%.
⦁ In Vitro Release of Encapsulated SB431542. In vitro release of SB431542 from SB-Lips was investigated using the dialysis method.21 1 mL of SB-Lips or free SB431542 (1 mg/mL of SB431542) was added into dialysis bags (Millipore, USA) with a molecular weight cut-oﬀ of 8−14 kDa. Dialysis bags were immersed in 4 mL release medium (1.0 M phosphate buﬀered solution containing 0.2% Tween 80, pH 7.4) at 37 °C and stirred at 70 rpm. At pre-established time points, aliquots were withdrawn and replaced with 4 mL fresh release medium. The
concentration of SB431542 in the removed medium was quantiﬁed by HPLC, as described in Section 2.2.
⦁ Cell Culture and Treatments. LX-2, a human stellate cell line, was kindly provided by Prof. Lieming Xu at Shuguang Hospital at the Shanghai University of Traditional Chinese Medicine. LX-2 was cultured in Dulbecco’s modiﬁed Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA). To test the eﬀect of SB-Lips on TGF-β signaling, the cells were exposed to TGF-β1 (2 ng/mL; PeproTech, USA) and SB-Lips for 48 h, then harvested for analysis.
⦁ Pharmacokinetics of SB-Lips in Rats. 10 male Sprague-Dawley rats were randomly divided into two groups, one of which was intravenously given free SB431542 in 30% PEG 400 and the other, SB-Lips in 5% glucose. In both cases, the dosage was 10 mg/kg. At predetermined time points, blood was sampled through the orbit (0.3 mL) into heparinized tubes. Samples were centrifuged at 6000 rpm for 10 min, and the resulting plasma (0.1 mL) was vortexed with 0.4 mL of methanol for 10 min to extract SB431542. After being centrifuged at 13,000 rpm for 10 min, the organic phase was evaporated at 37
°C for 2 h under the nitrogen stream in a pressured gas blowing concentrator (HGC-24A, Tianjin, China). The resulting residue was further dissolved in methanol and analyzed by HPLC, as described in Section 2.2. The pharmacokinetics parameters were calculated using DAS 2.0.
⦁ Biodistribution of DiD-Loaded Liposomes in a Mouse Model of Liver Fibrosis. A mouse model of hepatic ﬁbrosis was produced by injecting C57BL/6J mice twice per week with CCl4 (0.75 mL/kg, Sigma-Aldrich, St. Louis, USA) for 8 weeks. Animals were intravenously given DiD-Lips or free DiD dissolved in 5% DMSO glycol at a dosage of 40 μg/kg per mice. At 4 h after administration, mice were sacriﬁced, and the following tissues were collected: heart, liver, spleen, lung, kidney, and brains. Fluorescence was analyzed using the IVIS Spectrum system (Lumina 3, PerkinElmer, USA).
⦁ Therapeutic Effects of SB-Lips in Mice with CCl4- Induced Liver Fibrosis. C57BL/6J mice were injected with CCl4 (0.75 mL/kg) intraperitoneally twice per week for 4 weeks, as described. Control animals were injected with olive oil. Then, the mice were treated with saline, free SB431542, or SB-Lips every 3 days for 4 weeks, during which time the CCl4 treatment continued. The SB431542 dosage was 2 mg per kg. After treatment for 8 weeks, all mice were sacriﬁced, and serum and tissues were collected for further analysis.
For histology, liver tissues were sectioned at a thickness of 4 μm and stained with hematoxylin and eosin (H&E), Masson’s trichrome, or Sirius red to assess the severity of ﬁbrosis and inﬂammation. Images were captured using a microscope (Nikon, Tokyo, Japan). The hepatic hydroxyproline content was measured using a commercial kit (catalog no. A030-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions.
⦁ RT-PCR Analysis. Total RNA was extracted from LX-2 cells or liver tissues using the TRIzol reagent and then reverse- transcribed into complementary DNA using the iScript cDNA synthesis kit (TaKaRa, Kyoto, Japan). Real-time PCR was performed in a CFX96 Real-Time system (Bio-Rad, Hercules, CA, USA) by using the SYBR green-based assay. The primers are listed in the ⦁ Supporting⦁ ⦁ Information⦁ (Table S1).
⦁ Western Blot. Total protein from LX-2 cells or liver tissues was resolved using sodium dodecyl sulfate-polyacryla- mide gel electrophoresis, then transferred onto polyvinylidene

B https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

Figure 1. Characterization of SB431542-loaded liposomes (SB-Lips) in vitro. (A) Schematic illustration of SB-Lips. (B) Dynamic light scattering data for blank Lip and SB-Lips. (C) Transmission electron microscopy images of blank Lip and SB-Lips. Scale bar is 50 nm.
Table 1. Characteristics of Blank Lip and SB-Lips (n = 3, Mean ± SD)

size (nm) PDI zeta potential (mV) DL (%) EE (%)
blank lip SB-lip 66.97 ± 1.35
66.38 ± 0.58 0.25 ± 0.0059
0.22 ± 0.0062 −0.095 ± 0.42
10.60 ± 0.30
3.06 ± 0.05
97.90 ± 1.70

Figure 2. Stability of SB-Lips and the proﬁles of drug release. (A) Variations in particle size and PDI of SB-Lips at 4 and 25 °C in vitro. Data are mean ±
SD (n = 3). (B) Release of free SB and SB-Lips at 37 °C in PBS (pH 7.4) in the presence of 0.2% Tween-80. Data are mean ± SD (n = 3).

nitrocellulose ﬁlter membranes. Membranes were incubated with primary antibodies against a-SMA (Sigma, USA), Col1a1
(Boster, China), t-Smad3 (CST, USA), p-Smad3 (CST, USA), t-Akt (Boster, China), p-Akt (Santa Cruze, USA), and β-tubulin

Figure 3. SB-Lips inhibited the activation of LX-2 cells. (A) IC50 of free SB and SB-Lips in LX-2 was measured by the MTT assay. (B,C) mRNA expression of ﬁbrosis-related genes in LX-2 cells when treated with free SB and SB-Lips (200 nM) under TGF-β stimulation. *P < 0.05 vs the vehicle group, **P < 0.01 vs the vehicle group, #P < 0.05 vs the free SB group, and ##P < 0.01 vs the free SB group.

(Zhengneng, China). Immunoblots were visualized using the LI-COR Odyssey System (Lincoln, NE, USA). Band intensities were quantitated using Image Studio analysis software version
4.0 (LI-COR).
⦁ Statistical Analysis. All quantitative data were expressed as mean ± SEM values from triplicate measurements unless otherwise noted. Statistical analysis was performed using Prism 5 (GraphPad Software, USA). Diﬀerences among groups were evaluated for signiﬁcance using single-factor ANOVA, followed by student’s t-test. Statistical signiﬁcance is indicated as
*P < 0.05.
⦁ RESULTS
⦁ Characterization of SB-Lips. SB-Lips were prepared by a thin-ﬁlm hydration method, as previously described.21 The structure of SB-Lips is shown in Figure 1A. Dynamic light scattering indicated a size of 66.38 ± 0.58 nm and narrow size distribution (Figure 1B, upper and lower panels). Encapsulating SB431542 into the liposomes changed zeta potential from
−0.095 ± 0.42 mV to 10.60 ± 0.30 mV. The liposomes showed
D

an EE % of 97.9 ± 1.7% and DL % of 3.06 ± 0.05% for the drug (Table 1).
Transmission electron microscopy conﬁrmed that SB-Lips had the multi-chamber morphology and generally spherical shape typical for liposomes (Figure 1C, upper and lower panels). Standing at 4 or 25 °C for 7 days did not result in substantially larger particle size, polydispersity index (PDI), or DL capacity (Figure 2A and Supporting Information, Figure S1), indicating that SB-Lips were stable in 5% glucose. The liposomes supported sustained release of SB431542: approximately 70% of the encapsulated drug was released from dialysis bags after 72 h incubation, compared to over 90% of the free drug within 10 h (Figure 2B).
⦁ SB-Lips Inhibit HSC Activation via Modulating TGF-β/Smad Signaling. The proliferation and activation of HSCs is the central driver of liver ﬁbrosis,25,26 so we evaluated the eﬀects of SB-Lips on the human HSC cell line LX-2. MTT assays showed 50% inhibitory concentration (IC50) of 43.18 μM for free SB431542 and 5.51 μM for SB-Lips after 48-h incubation (Figure 3A, left and right panels). These results

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

Figure 4. SB-Lips inhibited the activation of LX-2 cells via modulating TGF-β/Smad signaling. (A) Protein levels of α-SMA and Col1a1. (B) Quantiﬁcation of western blot analysis in (A). The protein level was quantiﬁed by densitometry and normalized by β-tubulin. (C) Protein expression of p-Smad3 and p-Akt. (D) Quantiﬁcation of western blot analysis in (C). The protein level was quantiﬁed by densitometry and normalized by t-Smad3 or t-Akt. LX-2 cells were treated with and free SB or SB-Lips (200 nM) in the presence of TGF-β (2 ng/mL) for 48 h *P < 0.05.

Figure 5. In vivo pharmacokinetic behavior and distribution of SB-Lips. (A) Plasma concentration of SB431542 at diﬀerent time points after an i.v. injection of free SB and SB-Lips in rats. Data are shown as mean ± SD (n = 5). (B) Pharmacokinetic parameters of free SB and SB-Lips in rats. AUC, area under the concentration−time curve; t1/2z, half-life. Data are mean ± SD (n = 5). (C) Ex vivo images of major organs 4 h after being treated with free DiD and DiD-Lips. (D) Semiquantitative ﬂuorescence intensity of liver. Data are mean ± SD (n = 3).

suggested that SB-Lips were much more eﬀective than the free drug at inhibiting HSC proliferation. Under this condition, free and SB-Lips had no toxicity on normal hepatocytes (Supporting Information, Figure S2A).

Next, we compared the inhibitory ability of the free drug or SB-Lips on HSC activation, which depends on TGF-β signaling. As expected, treating LX-2 cells with 2 ng/mL TGF-β1 upregulated transcriptions of the ﬁbrotic genes (α-SMA, Col1a1, Col1a2, and TGF-β) and matrix metalloprotease

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

Figure 6. SB-Lips alleviated hepatic ﬁbrosis in the CCl4-induced mouse model. (A) Regimen of treatment of free SB or SB-Lip. C57BL/6J mice were injected with olive oil or CCl4 (0.75 mL/kg) for 8 weeks. In the last 4 weeks, mice received tail-vein injection of free SB or SB-Lips (2 mg/kg) every 3 days. (B) Representative images of histological staining demonstrate the liver injury, collagen deposition in the indicated groups. (C) Concentration of hydroxyproline in the liver. (D,E) Hepatic mRNA expression of ﬁbrotic genes such as α-SMA, Col1a1, and Col1a2 (D); Col3a1, Timp-1, MMP-9, and TGF-β (E). *P < 0.05 and **P < 0.01 vs the vehicle group, #P < 0.05, ##P < 0.01, and ###P < 0.001 vs the free SB group.

(MMP)-2. This upregulation was substantially dampened by SB-Lips, whereas the same concentration of free SB531542 did
not obviously alter transcript levels of ﬁbrotic genes (Figure 3B,C). Consistent with mRNA expression, treating LX-2 cells

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

Figure 7. SB-Lips alleviated CCl4-induced liver ﬁbrosis by suppressing the TGF-β/Smad pathway. (A) Protein levels of α-SMA and Col1a1. (B) Quantiﬁcation of western blot analysis in (A). The protein level was quantiﬁed by densitometry and normalized by β-tubulin. (C) Protein expression of TGF-β and p-Smad3. (D) Quantiﬁcation of western blot analysis in (C). The protein level was quantiﬁed by densitometry and normalized by β-tubulin or t-Smad3. *P < 0.05, **P < 0.01, and ***P < 0.001 vs the vehicle group, #P < 0.05, ##P < 0.01, and ###P < 0.001 vs the free SB group.

with SB-Lips led to the most signiﬁcant reduced protein expressions of α-SMA and Col1a1 (Figure 4A,B). Under the inhibitory dose, SB431542 treatment did not inﬂuence the cell apoptosis/necrosis pathway (Supporting Information, Figure S3A).
To verify the enhanced ability of SB-Lips to inhibit TGF-β signaling, we compared the inhibitory eﬀect of the free drug and SB-Lips on the phosphorylation of Smad2 and Smad3, the downstream eﬀectors.27 SB-Lips signiﬁcantly abolished the TGF-β1-induced phosphorylation of Smad3, while the free drug only showed little eﬀect (Figure 4C,D). Neither treatment, however, aﬀected total levels of Smad3. As SB431542 is a synthetic small molecule that speciﬁcally inhibits TGF-β signaling mediated by ALK4, ALK5, or ALK7,15 we detected the ALK4, ALK5, and ALK7 mRNA levels both in vivo and in vitro (Supporting Information, Figure S3B,C). The results showed that SB431542 treatment can only decrease ALK5 expression but not inﬂuence ALK4 and ALK7 expression under the treatment dose.
To further explore the antiproliferative eﬀect of SB-Lips, we examined Akt phosphorylation, which is important in HSC proliferation.28 TGF-β1 slightly promoted Akt phosphorylation, which SB-Lips reversed to a greater extent than the free drug (Figure 4C,D).
⦁ Pharmacokinetics of SB-Lips in Rats. To assess whether SB-Lips were able to keep the drug in circulation for longer time in vivo, we compared the pharmacokinetics of free SB431542 and SB-Lips in male Sprague-Dawley rats. During the ﬁrst 90 min after the intravenous injection, signiﬁcantly more drug was present in the circulation of animals treated with SB- Lips than in the circulation of animals treated with the free drug (Figure 5A). By 4 h after the injection, the level of drugs in both
groups was nearly undetectable. The SB-Lips group exhibited much higher area under the curve (AUC0‑t) and Cmax (both P < 0.001) compared with the free drug group, and the drug’s half- life (t1/2) was slightly longer in the SB-Lips group (Figure 5B).

⦁ Biodistribution of DiD-Lips in CCl4-Induced Fibrotic Mouse Model. For biodistribution studies in the CCl4-induced ﬁbrotic model, we used liposomes containing the ﬂuorescent dye DiD instead of SB431542. Healthy control mice showed similar distributions of free DiD or encapsulated DiD, with negligible ﬂuorescent signal in all collected organs (Figure 5C,D). Fibrotic mice showed a similar ﬂuorescence distribution to healthy mice after the injection of free DiD, whereas DiD accumulated in the liver after the injection of DiD-Lips.
⦁ SB-Lips Alleviate Hepatic Fibrosis by Inhibiting TGF-β/Smad Pathway in CCl4-Induced Fibrotic Model. To evaluate the therapeutic potential of SB-Lips for liver ﬁbrosis in vivo, we injected mice intraperitoneally with 0.75 mL/kg CCl4 for 8 weeks.27 The regimen of drug treatment in the CCl4- induced animal model is illustrated in Figure 6A. CCl4 treatment destroyed the liver tissue structure and led to collagen accumulation and inﬂammatory cell inﬁltration (Figure 6B). SB-Lips alleviated collagen accumulation to a much greater extent than free SB431542, based on tissue staining (Figure 6B and ⦁ Supporting Information, Figure S4A) and hydroxyproline assays (Figure 6C) without aﬀecting inﬂammatory cell inﬁltration and liver injury (Figure 6B and ⦁ Supporting ⦁ Information, Figure S4B,C).
The CCl4 injection upregulated the mRNA expression of α- SMA, collagen, Timp-1, MMP-9, and TGF-β. SB-Lips showed more pronounced inhibitory eﬀects on these ﬁbrotic genes than the free drug (Figure 6D,E). Consistent with these transcrip- tional results, SB-Lips strongly reduced the CCl4-increased protein expression of α-SMA and Col1a1 (Figure 7A,B). In addition, SB-Lips were more eﬀective than the free drug in inhibiting CCl4-induced phosphorylation of Smad3 (Figure 7C,D). Interestingly, SB-Lips also reduced the protein expression of TGF-β (Figure 7C,D). Either free SB or SB-Lips treatment had no inﬂuence on liver ﬁbrosis, inﬂammation, and histological tissue damage under olive treatment (Supporting Information, Figures S5−S7).
G https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

Figure 8. Safety evaluation of SB-Lips in mice. The representative images of H&E staining demonstrate injury of the major organs in the indicated groups.

⦁ Safety Evaluation of SB-Lips in Mice. The potentially wide-ranging systemic eﬀects of TGF-β signaling made us concerned about whether SB-Lips might cause toxic eﬀects in oﬀ-target organs, including the heart, spleen, lung, and kidney. H&E staining showed that mice treated with SB-Lips showed no damage among the organs examined (Figure 8).

⦁ DISCUSSION
SB431542, a speciﬁc inhibitor of TGF-β1 receptor kinase, has shown potential for the treatment of ﬁbrosis-related diseases, but physical and chemical properties limit its clinical applica- tion.29−31 SB431542 inhibits ALK5 potently and selectively (>100-fold) over many other kinases, allowing it to block TGF- β1-induced upregulation of ﬁbronectin, PAI-1, and Col1a1,15 which is also conﬁrmed in the Supporting Information (Figure S3B,C). Here, we exploited the ability of nanoparticles such as liposomes to accumulate in well-vascularized organs32 in order to construct a delivery system that appears to be superior to free SB431542 for ensuring persistence of the drug in circulation and for exerting antiﬁbrotic eﬀects on the liver through inhibition of TGF-β and PI3K/Akt signaling pathways.
In the present study, we loaded SB431542 into a liposome, which inhibited HSC proliferation and activation through TGF- β and PI3K/Akt pathway inhibition. Upon the dosage of 200 nmol/L used in in vitro study, SB-Lips appeared to have little eﬀect on the apoptosis of HSCs. Neither SB-Lips had toxicity on the hepatocyte, as demonstrated by the MTT assay. SB-Lips showed a potential for the treatment of liver ﬁbrosis in the CCl4- induced ﬁbrosis mouse model but did not aﬀect liver injury and
inﬂammation. This may be because SB mainly aﬀects the ECM secreted by the activated HSCs.33
To our knowledge, this is the ﬁrst nanoformulation of SB431542 that demonstrates its therapeutic potential against liver ﬁbrosis. SB431542 is water insoluble, and it shows poor solubility in acetone, chloroform, dichloromethane, and other organic solvents commonly used to prepare nanoparticles. To improve solubility, we ﬁrst formed a complex of the drug with unsaturated OA, which has been shown to increase the liposolubility of α-lactoalbumin.34 Then, we loaded this complex into liposomes based on S100 and the surfactant Solutol HS15. Solutol HS15 is a new type of PEG series that shows lower toxicity, higher stability, and lower cost than other frequently used excipients in long-circulation liposomes, such as DSPE- PEG2000.21,35−37 The solubilizing eﬀects of our liposomes may help explain why the encapsulated drug showed signiﬁcantly greater Cmax and higher AUC than the free drug. The liposomes are known to overcome the obstacle of the cellular uptake of compounds,38 and this property may explain the enhanced eﬃcacy of SB-Lips on proliferation and activation of HSCs in vitro.
Most drugs against hepatic ﬁbrosis target one or few
molecules, such as pirfenidone, which targets TGF-β1; sorafenib, which targets receptors for the platelet-derived growth factor or vascular endothelial growth factor; and obeticholic acid, which targets the farnesoid X receptor.39−41 These drugs often cause obvious adverse reactions due to high dosage to ensure suﬃciently therapeutic eﬃcacy against the background of pleiotropic eﬀects of their target molecules. They also show poor biodistribution and pharmacokinetics.32 Treating liver ﬁbrosis eﬀectively therefore requires targeting

H https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

appropriate molecules selectively in aﬀected tissues, which our SB-Lips appear to do.
One factor that may promote accumulation of SB-Lips in ﬁbrotic tissues is the presence of damaged blood vessels. The vascular proliferation in ﬁbrotic liver increases vascular permeability, which may result in diminished blood ﬂow through the organ, analogous to the situation with solid tumors.18−20,42 The passive targeting of SB-Lips to ﬁbrotic tissues not only enhances their therapeutic eﬀects but also reduces their distribution to other tissues, which may help explain why we observed no obvious toxic eﬀects on other key organs. This ability to target ﬁbrotic tissues may make SB-Lips useful in other diseases that involve ﬁbrosis, such as renal ﬁbrosis and lung ﬁbrosis.
⦁ CONCLUSIONS
■
Loading the water-insoluble drug SB431542 into liposomes based on S100 and Solutol HS15 greatly increased its solubility and improved pharmacokinetic properties after the intravenous injection. These stable and uniform nanoparticles showed better eﬃcacy than free SB431542 in alleviating hepatic ﬁbrosis in vitro and in vivo through the inhibition of TGF-β1 signaling. SB-Lips improved the drug’s therapeutic eﬃcacy without damaging oﬀ- target tissues. These results suggest therapeutic potential of SB- Lips against liver ﬁbrosis.
ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharma- ceut.0c00633.
Isolation methods of hepatocytes, therapeutic eﬃcacy, and primers and antibody lists (PDF)
⦁ AUTHOR INFORMATION
Corresponding Authors
Yanping Li − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China; Phone: 86-28- 85426416; Email: [email protected]
Jinhan He − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China; orcid.org/ 0000-0001-6487-4696; Phone: 86-28-85426416;
Email: [email protected]
Authors
Jinhang Zhang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Rui Li − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Qinhui Liu − Laboratory of Clinical Pharmacy and Adverse Drug Reaction, West China Hospital of Sichuan University, Chengdu 610041, China
Jian Zhou − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Hui Huang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Ya Huang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Zijing Zhang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Tong Wu − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Qin Tang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Cuiyuan Huang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Yingnan Zhao − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Guorong Zhang − Laboratory of Clinical Pharmacy and Adverse Drug Reaction and Department of Pharmacy, West China Hospital of Sichuan University, Chengdu 610041, China
Li Mo − Center of Gerontology and Geriatrics, West China Hospital of Sichuan University, Chengdu 610041, China
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00633

Notes
■
The authors declare no competing ﬁnancial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science Foundation of China (81870599, 81873662, and 81930020), research funding from Sichuan Province (2020YFS0064), Post- Doctor Research Project, and West China Hospital of Sichuan University (2018HXBH060). The authors thank Huifang Li and Yan Wang from the Core Facility of West China Hospital, Li and Fei Chen from the Laboratory of Pathology, West China Hospital for technical assistance.
ABBREVIATIONSPARA
■
α-SMA, α-smooth muscle actin; Col, collagen; CCl4, carbon tetrachloride; DiD, 1,10-dioctadecyl- 3,3,30,30- tetramethylin- dodicarbocyanine, 4-chlorobenzenesulfonate salt; ECM, extrac- ellular matrix; DLS, dynamic light scattering; EPR eﬀect, enhanced permeability and retention eﬀect; FBS, fetal bovine serum; HSC, hepatic stellate cell; MMP2, matrix metal- loprotease-2 PDI polydispersity index; OA, oleic acid; SB- Lips, liposomes loaded with SB431542; TGF-β, transforming growth factor-β
REFERENCES
⦁ Friedman, S. L. ⦁ Liver⦁ ⦁ fibrosis⦁ ⦁ −⦁ ⦁ from⦁ ⦁ bench⦁ ⦁ to⦁ ⦁ bedside.⦁ J. Hepatol.
2003, 38, 38−53.
⦁ Schuppan, D.; Ashfaq-Khan, M.; Yang, A. T.; Kim, Y. O. ⦁ Liver ⦁ fibrosis:⦁ ⦁ Direct⦁ ⦁ antifibrotic⦁ ⦁ agents⦁ ⦁ and⦁ ⦁ targeted⦁ ⦁ therapies.⦁ Matrix Biol. 2018, 68−69, 435−451.
⦁ Bataller, R.; Brenner, D. A. ⦁ Liver⦁ ⦁ fibrosis.⦁ J. Clin. Invest. 2005, 115, 209−218.
⦁ Sun, M.; Kisseleva, T. ⦁ Reversibility⦁ ⦁ of⦁ ⦁ liver⦁ ⦁ fibrosis. Clin. Res. Hepatol. Gastroenterol. 2015, 39, S60−S63.
⦁ Seki, E.; Brenner, D. A. ⦁ Recent advancement of molecular ⦁ mechanisms⦁ ⦁ of⦁ ⦁ liver⦁ ⦁ fibrosis.⦁ J. Hepato-Biliary-Pancreatic Sci. 2015, 22, 512−518.

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

⦁ Mormone, E.; George, J.; Nieto, N. ⦁ Molecular pathogenesis ⦁ of ⦁ hepatic fibrosis and current therapeutic approaches. Chem.-Biol. Interact. 2011, 193, 225−231.
⦁ Tsuchida, T.; Friedman, S. L. ⦁ Mechanisms⦁ ⦁ of⦁ ⦁ hepatic⦁ ⦁ stellate⦁ ⦁ cell ⦁ activation.⦁ Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 397−411.
⦁ Friedman, S. L. ⦁ Hepatic⦁ ⦁ stellate⦁ ⦁ cells:⦁ ⦁ protean,⦁ ⦁ multifunctional,⦁ ⦁ and ⦁ enigmatic⦁ ⦁ cells⦁ ⦁ of⦁ ⦁ the⦁ ⦁ liver.⦁ Physiol. Rev. 2008, 88, 125−172.
⦁ Li, Y.; Pu, S.; Liu, Q.; Li, R.; Zhang, J.; Wu, T.; Chen, L.; Li, H.; Yang, X.; Zou, M.; Xiao, J.; Xie, W.; He, J. ⦁ An integrin-based ⦁ nanoparticle⦁ ⦁ that⦁ ⦁ targets⦁ ⦁ activated⦁ ⦁ hepatic⦁ ⦁ stellate⦁ ⦁ cells⦁ ⦁ and⦁ ⦁ alleviates ⦁ liver fibrosis. J. Controlled Release 2019, 303, 77−90.
⦁ Gressner, A. M.; Gressner, R. W.; Breitkopf, K.; Dooluy, S. ⦁ Roles ⦁ of⦁ ⦁ TGFbeta⦁ ⦁ in⦁ ⦁ hepatic⦁ ⦁ fibrosis.⦁ Front. Biosci. 2002, 7, d793−807.
⦁ Caja, L.; Dituri, F.; Mancarella, S.; Caballero-Diaz, D.; Moustakas, A.; Giannelli, G.; Fabregat, I. ⦁ TGF-beta⦁ ⦁ and⦁ ⦁ the⦁ ⦁ Tissue ⦁ Microenvironment:⦁ ⦁ Relevance⦁ ⦁ in⦁ ⦁ Fibrosis⦁ ⦁ and⦁ ⦁ Cancer.⦁ Int. J. Mol. Sci. 2018, 19, 1294.
⦁ Heldin, C.-H.; Moustakas, A. ⦁ Role of Smads in TGFbeta ⦁ signaling. Cell Tissue Res. 2012, 347, 21−36.
⦁ Miyazawa, K.; Miyazono, K. ⦁ Regulation of TGF-beta ⦁ Family ⦁ Signaling⦁ ⦁ by⦁ ⦁ Inhibitory⦁ ⦁ Smads.⦁ Cold Spring Harbor Perspect. Biol. 2017, 9, a022095.
⦁ Su, K.-Y.; Hsieh, C.-Y.; Chen, Y.-W.; Chuang, C.-T.; Chen, C.-T.; Chen, Y.-L. S. ⦁ Taiwanese⦁ ⦁ Green⦁ ⦁ Propolis⦁ ⦁ and⦁ ⦁ Propolin⦁ ⦁ G⦁ ⦁ Protect⦁ ⦁ the ⦁ Liver⦁ ⦁ from⦁ ⦁ the⦁ ⦁ Pathogenesis⦁ ⦁ of⦁ ⦁ Fibrosis⦁ ⦁ via⦁ ⦁ Eliminating⦁ ⦁ TGF-⦁ β⦁ -Induced ⦁ Smad2/3 Phosphorylation. J. Agric. Food Chem. 2014, 62, 3192−3201.
⦁ Inman, G. J.; Nicolaś, F. J.; Callahan, J. F.; Harling, J. D.; Gaster,
L. M.; Reith, A. D.; Laping, N. J.; Hill, C. S. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 2002, 62, 65−74.
⦁ Waghabi, M. C.; Keramidas, M.; Calvet, C. M.; Meuser, M.; Soeiro, M. d. N. C.; Mendonca̧-Lima, L.; Arauj́o-Jorge, T. C.; Feige, J.- J.; Bailly, S. ⦁ SB-431542, a transforming growth factor beta inhibitor, ⦁ impairs Trypanosoma cruzi infection in cardiomyocytes and parasite ⦁ cycle⦁ ⦁ completion.⦁ Antimicrob. Agents Chemother. 2007, 51, 2905−2910.
⦁ Jimeńez Calvente, C.; Sehgal, A.; Popov, Y.; Kim, Y. O.; Zevallos, V.; Sahin, U.; Diken, M.; Schuppan, D. ⦁ Specific⦁ ⦁ hepatic⦁ ⦁ delivery⦁ ⦁ of ⦁ procollagen alpha1(I) small interfering RNA in lipid-like ⦁ nanoparticles ⦁ resolves liver fibrosis. Hepatology 2015, 62, 1285−1297.
⦁ Zhang, S.; Wu, J.; Wang, H.; Wang, T.; Jin, L.; Shu, D.; Shan, W.;
⦁ Kinoshita, K.; Iimuro, Y.; Fujimoto, J.; Inagaki, Y.; Namikawa, K.; Kiyama, H.; Nakajima, Y.; Otogawa, K.; Kawada, N.; Friedman, S. L.; Ikeda, K. ⦁ Targeted⦁ ⦁ and⦁ ⦁ regulable⦁ ⦁ expression⦁ ⦁ of⦁ ⦁ transgenes⦁ ⦁ in⦁ ⦁ hepatic ⦁ stellate cells and myofibroblasts in culture and in vivo using ⦁ an ⦁ adenoviral⦁ ⦁ Cre/lo⦁ x⦁ P⦁ ⦁ system⦁ ⦁ to⦁ ⦁ antagonise⦁ ⦁ hepatic⦁ ⦁ fibrosis.⦁ Gut 2007, 56, 396−404.
⦁ Trautwein, C.; Friedman, S. L.; Schuppan, D.; Pinzani, M. ⦁ Hepatic⦁ ⦁ fibrosis:⦁ ⦁ Concept⦁ ⦁ to⦁ ⦁ treatment.⦁ J. Hepatol. 2015, 62, S15−S24.
⦁ Dufton, N. P.; Peghaire, C. R.; Osuna-Almagro, L.; Raimondi, C.; Kalna, V.; Chuahan, A.; Webb, G.; Yang, Y.; Birdsey, G. M.; Lalor, P.; Mason, J. C.; Adams, D. H.; Randi, A. M. ⦁ Dynamic regulation⦁ ⦁ of ⦁ canonical⦁ ⦁ TGFbeta⦁ ⦁ signalling⦁ ⦁ by⦁ ⦁ endothelial⦁ ⦁ transcription⦁ ⦁ factor⦁ ⦁ ERG ⦁ protects⦁ ⦁ from⦁ ⦁ liver⦁ ⦁ fibrogenesis.⦁ Nat. Commun. 2017, 8, 895.
⦁ Han, C. Y.; Koo, J. H.; Kim, S. H.; Gardenghi, S.; Rivella, S.; Strnad, P.; Hwang, S. J.; Kim, S. G. ⦁ Hepcidin inhibits Smad3 ⦁ phosphorylation in hepatic stellate cells by impeding ⦁ ferroportin- ⦁ mediated⦁ ⦁ regulation⦁ ⦁ of⦁ ⦁ Akt.⦁ Nat. Commun. 2016, 7, 13817.
⦁ Laping, N. J.; Grygielko, E.; Mathur, A.; Butter, S.; Bomberger, J.; Tweed, C.; Martin, W.; Fornwald, J.; Lehr, R.; Harling, J.; Gaster, L.; Callahan, J. F.; Olson, B. A. ⦁ Inhibition⦁ ⦁ of⦁ ⦁ Transforming⦁ ⦁ Growth⦁ ⦁ Factor ⦁ (TGF)-beta1⦁ −⦁ Induced⦁ ⦁ E⦁ x⦁ tracellular⦁ ⦁ Matri⦁ x⦁ ⦁ with⦁ ⦁ a⦁ ⦁ Novel⦁ ⦁ Inhibitor⦁ of ⦁ the TGF-beta Type I Receptor Kinase Activity: SB-431542. Mol. Pharmacol. 2002, 62, 58.
⦁ Bonniaud, P.; Margetts, P. J.; Kolb, M.; Schroeder, J. A.; Kapoun,
A. M.; Damm, D.; Murphy, A.; Chakravarty, S.; Dugar, S.; Higgins, L.; Protter, A. A.; Gauldie, J. Progressive transforming growth factor beta1- induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am. J. Respir. Crit. Care Med. 2005, 171, 889−898.
⦁ de Gouville, A.-C.; Boullay, V.; Krysa, G.; Pilot, J.; Brusq, J.-M.; Loriolle, F.; Gauthier, J.-M.; Papworth, S. A.; Laroze, A.; Gellibert, F.; Huet, S. ⦁ Inhibition⦁ ⦁ of⦁ ⦁ TGF-beta⦁ ⦁ signaling⦁ ⦁ by⦁ ⦁ an⦁ ⦁ ALK5⦁ ⦁ inhibitor⦁ ⦁ protects ⦁ rats from dimethylnitrosamine-induced liver fibrosis. Br. J. Pharmacol. 2005, 145, 166−177.
⦁ Tee, J. K.; Peng, F.; Ho, H. K. ⦁ Effects⦁ ⦁ of⦁ ⦁ inorganic⦁ ⦁ nanoparticles ⦁ on⦁ ⦁ liver⦁ ⦁ fibrosis:⦁ ⦁ Optimizing⦁ ⦁ a⦁ ⦁ double-edged⦁ ⦁ sword⦁ ⦁ for⦁ ⦁ therapeutics. Biochem. Pharmacol. 2019, 160, 24−33.
⦁ Zhang, S.; Sun, W.-Y.; Wu, J.-J.; Wei, W. ⦁ TGF-⦁ β ⦁ signaling ⦁ pathway⦁ ⦁ as⦁ ⦁ a⦁ ⦁ pharmacological⦁ ⦁ target⦁ ⦁ in⦁ ⦁ liver⦁ ⦁ diseases.⦁ Pharmacol. Res. 2014, 85, 15−22.
⦁ Meikle, V.; Mossberg, A.-K.; Mitra, A.; Hakansson, A. P.; Niederweis, M. ⦁ A⦁ ⦁ Protein⦁ ⦁ Complex⦁ ⦁ from⦁ ⦁ Human⦁ ⦁ Milk⦁ ⦁ Enhances⦁ ⦁ the

Xiong, S. Liposomal oxymatrine
in hepatic fibrosis treatment:
Activity of Antibiotics and Drugs against Mycobacterium tuberculosis.

formulation, in vitro and in vivo assessment. AAPS PharmSciTech
2014, 15, 620−629.
⦁ DeLeve, L. ⦁ Hepatic microvasculature in liver injury. Semin. Liver Dis. 2007, 27, 390−400.
⦁ Yoshiji, H.; Kuriyama, S.; Yoshii, J.; Ikenaka, Y.; Noguchi, R.; Hicklin, D. J.; Wu, Y.; Yanase, K.; Namisaki, T.; Yamazaki, M.; Tsujinoue, H.; Imazu, H.; Masaki, T.; Fukui, H. ⦁ Vascular⦁ ⦁ endothelial ⦁ growth factor and receptor interaction is a prerequisite for ⦁ murine ⦁ hepatic fibrogenesis. Gut 2003, 52, 1347−1354.
⦁ Jia, M.; Deng, C.; Luo, J.; Zhang, P.; Sun, X.; Zhang, Z.; Gong, T. ⦁ A⦁ ⦁ novel⦁ ⦁ dexamethasone-loaded⦁ ⦁ liposome⦁ ⦁ alleviates⦁ ⦁ rheumatoid⦁ ⦁ arthritis ⦁ in rats. Int. J. Pharm. 2018, 540, 57−64.
⦁ Hofkens, W.; Grevers, L. C.; Walgreen, B.; de Vries, T. J.; Leenen,
P. J. M.; Everts, V.; Storm, G.; van den Berg, W. B.; van Lent, P. L. Intravenously delivered glucocorticoid liposomes inhibit osteoclast activity and bone erosion in murine antigen-induced arthritis. J. Controlled Release 2011, 152, 363−369.
⦁ Li, R.; Li, Y.; Zhang, J.; Liu, Q.; Wu, T.; Zhou, J.; Huang, H.; Tang, Q.; Huang, C.; Huang, Y.; Zhang, Z.; Zhang, G.; Zhao, Y.; Ma, L.; Feng, Y.; Mo, L.; Han, M.; He, J. ⦁ Targeted⦁ ⦁ delivery⦁ ⦁ of⦁ ⦁ celastrol⦁ ⦁ to⦁ ⦁ renal ⦁ interstitial myofibroblasts using fibronectin-binding liposomes ⦁ attenu- ⦁ ates⦁ ⦁ renal⦁ ⦁ fibrosis⦁ ⦁ and⦁ ⦁ reduces⦁ ⦁ systemic⦁ ⦁ to⦁ x⦁ icity.⦁ J. Controlled Release 2020, 320, 32−44.
⦁ Li, Y.; Liu, Q.; Li, W.; Zhang, T.; Li, H.; Li, R.; Chen, L.; Pu, S.; Kuang, J.; Su, Z.; Zhang, Z.; He, J. ⦁ Design⦁ ⦁ and⦁ ⦁ Validation⦁ ⦁ of⦁ ⦁ PEG- ⦁ Derivatized⦁ ⦁ Vitamin⦁ ⦁ E⦁ ⦁ Copolymer⦁ ⦁ for⦁ ⦁ Drug⦁ ⦁ Delivery⦁ ⦁ into⦁ ⦁ Breast ⦁ Cancer. Bioconjugate Chem. 2016, 27, 1889−1899.
Antimicrob. Agents Chemother. 2018, 63, No. e01846.
⦁ Li, X.; Zhang, Y.; Fan, Y.; Zhou, Y.; Wang, X.; Fan, C.; Liu, Y.; Zhang, Q. ⦁ Preparation⦁ ⦁ and⦁ ⦁ evaluation⦁ ⦁ of⦁ ⦁ novel⦁ ⦁ mi⦁ x⦁ ed⦁ ⦁ micelles⦁ ⦁ as ⦁ nanocarriers⦁ ⦁ for⦁ ⦁ intravenous⦁ ⦁ delivery⦁ ⦁ of⦁ ⦁ propofol.⦁ Nanoscale Res. Lett. 2011, 6, 275.
⦁ Alani, A. W. G.; Rao, D. A.; Seidel, R.; Wang, J.; Jiao, J.; Kwon, G.
S. The effect of novel surfactants and Solutol HS 15 on paclitaxel aqueous solubility and permeability across a Caco-2 monolayer. J. Pharm. Sci. 2010, 99, 3473−3485.
⦁ Lu, H.; Li, J.; Li, M.; Gong, T.; Zhang, Z. ⦁ Systemic⦁ ⦁ delivery⦁ ⦁ of ⦁ alpha-asarone⦁ ⦁ with⦁ ⦁ Kolliphor⦁ ⦁ HS⦁ ⦁ 15⦁ ⦁ improves⦁ ⦁ its⦁ ⦁ safety⦁ ⦁ and⦁ ⦁ therapeutic ⦁ effect on asthma. Drug Deliv. 2015, 22, 266−275.
⦁ Sercombe, L.; Veerati, T.; Moheimani, F.; Wu, S. Y.; Sood, A. K.; Hua, S. ⦁ Advances⦁ ⦁ and⦁ ⦁ Challenges⦁ ⦁ of⦁ ⦁ Liposome⦁ ⦁ Assisted⦁ ⦁ Drug⦁ ⦁ Delivery. Front. Pharmacol. 2015, 6, 286.
⦁ Flores-Contreras, L.; Sandoval-Rodríguez, A. S.; Mena-Enriquez,
M. G.; Lucano-Landeros, S.; Arellano-Olivera, I.; Álvarez-Álvarez, A.; Sanchez-Parada, M. G.; Armendaŕiz-Borunda, J. Treatment with pirfenidone for two years decreases fibrosis, cytokine levels and
enhances CB2 gene expression in patients with chronic hepatitis C.
BMC Gastroenterol. 2014, 14, 131.
⦁ Cannito, S.; Novo, E.; Parola, M. ⦁ Therapeutic pro-fibrogenic ⦁ signaling⦁ ⦁ pathways⦁ ⦁ in⦁ ⦁ fibroblasts.⦁ Adv. Drug Deliv. Rev. 2017, 121, 57− 84.
⦁ Ma, R.; Chen, J.; Liang, Y.; Lin, S.; Zhu, L.; Liang, X.; Cai, X. ⦁ Sorafenib: A potential therapeutic drug for hepatic fibrosis and ⦁ its ⦁ outcomes.⦁ Biomed. Pharmacother. 2017, 88, 459−468.

https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633

⦁ Fernańdez, M.; Semela, D.; Bruix, J.; Colle, I.; Pinzani, M.; Bosch,
J. Angiogenesis in liver disease. J. Hepatol. 2009, 50, 604−620.

K https://dx.doi.org/10.1021/acs.molpharmaceut.0c00633