Development of 11-DGA-3-O-Gal-Modified Cantharidin Liposomes for Treatment of Hepatocellular Carcinoma

Background: Liver cancer is a common malignant tumor worldwide, and its morbidity and mortality increase each year. The disease has a short course and high mortality, making it a serious threat to human health. Purpose: The objective of this study was to create novel liver-targeting nanoliposomes to encapsulate cantharidin (CTD) as a potential treatment for hepatic carcinoma. Methods: 3-Galactosidase-30-stearyl deoxyglycyrrhetinic acid (11-DGA-3-O-Gal)-modified liposomes (11-DGA-3-O-Gal-CTD-lip) for the liver-targeted delivery of CTD were prepared via the film-dispersion method and characterized. In vitro analyses of the effects on cellular cytotoxicity, cell migration, cell cycle, and cell apoptosis were carried out and an in vivo pharmacokinetics study and tissue distribution analysis were performed. Results: Compared with unmodified liposomes (CTD-lip), 11-DGA-3-O-Gal-CTD-lip showed higher cytotoxicity and increased the inhibition of HepG2 cell migration, but they did not increase the apoptotic rate of cells. The inhibition mechanism of 11-DGA-3-O-Gal-CTD-lip on hepatocellular carcinoma was partly through cell cycle arrest at the S phase. Analysis of pharmacokinetic parameters indicated that 11-DGA-3-O-Gal-CTD-lip were eliminated more rapidly than CTD-lip. Regarding tissue distribution, the targeting efficiency of 11-DGA-3-O-Gal-CTD-lip to the liver was (41.15 ± 3.28)%, relative targeting efficiency was (1.53 ± 0.31)%, relative uptake rate was( 1.69 ± 0.37)%, and peak concentration ratio was (2.68 ± 0.12)%. Conclusion: 11-DGA-3-O-Gal-CTD-lip represent a promising nanocarrier for the liver-targeted delivery of antitumor drugs to treat hepatocellular carcinoma.


Introduction
As a major public health concern worldwide, cancer has received widespread attention from all parts of society, but the burden of cancer will increase in coming decades, especially in low-and middle-income countries (LMIC) [1,2]. Although the fight against cancer has been continuous, the mortality rate for various cancers has only decreased by 2% [3]. Primary liver cancer, comprised majorly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma, had become the 5th most common malignant tumor, the 2th cause of cancer death worldwide in 2018 [4]. HCC is one of the most common malignant tumors worldwide, and its morbidity and mortality increase each year [5,6]. HCC has a short course of disease and high mortality, presenting a serious threat to human health. Chemotherapy and surgery remain the main treatments to date [7]. Most anti-cancer drugs used for cancer treatment are highly toxic and have poor specificity [8]. Moreover, tumor cells are prone to developing resistance to chemotherapeutic drugs, which also leads to poor therapeutic effects [9]. Therefore, there is an urgent need to develop a new treatment plan for liver cancer.

Synthesis and Structure Confirmation of 11-DGA-3-O-Gal
11-DGA-3-O-Gal was synthesized successfully by esterification reaction in organic media. Both synthesis steps were monitored by thin-layer chromatography (TLC) to ensure complete the reaction. The molecular weight detected was 893.69 [M + Na] + in positive-ion mode, which suggested that the product obtained was identical with 11-DGA-3-O-Gal (molecular weight: 870.69). The 13 C-nuclear magnetic resonance (NMR) spectrum of 3-acetylation-galactosidase-30-stearyl deoxyglycyrrhetinic acid showed that the chemical shift values at the C3 atom was shifted to a lower magnetic field (δ79.03→δ90.92), which indicated esterification proceeded between DGA and acetobromo-α-d-galactose.   Table 1. Sizes, polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE) of different types of liposomes (n = 3).

Characterization of Liposomes
Both types of liposomes (11-DGA-3-O-Gal-CTD-lip and CTD-lip) were prepared using the film dispersion method (Figure 2). Visual examinations revealed that the lipid films were uniform and delicate, and the liposome solution obtained appeared clear and transparent with light blue opalescence. The size distribution, polydispersity index (PDI), and zeta potential (ZP) of the different liposomes are shown in Table 1. All liposomes had a narrow size distribution and relatively high entrapment efficiency. Images of liposome morphology were obtained by transmission electron microscopy. A single factor test was designed based on the appearance, particle size, and encapsulation efficiency of the liposomes. The ratio of targeting molecule-lipid (1:15, 1:10, 1:7.5, 1:6) added to the cantharidin liposome solution was investigated. As the amount of targeting molecule increased, the turbidity and particle size of liposomes increased. In the range of a single factor investigation, when the ratio was 1:10, the liposomes were clear and transparent with pale blue opalescence and a stable particle size, and the encapsulation efficiency was greater than 90%. No significant changes were observed in any characteristic following the addition of 11-DGA-3-O-Gal to the liposomes. The appearance, morphology, size distribution, and zeta potential distribution are presented in Figure 3.
Molecules 2019, 24, x FOR PEER REVIEW 4 of 18 entrapment efficiency. Images of liposome morphology were obtained by transmission electron microscopy. A single factor test was designed based on the appearance, particle size, and encapsulation efficiency of the liposomes. The ratio of targeting molecule-lipid (1:15, 1:10, 1:7.5, 1:6) added to the cantharidin liposome solution was investigated. As the amount of targeting molecule increased, the turbidity and particle size of liposomes increased. In the range of a single factor investigation, when the ratio was 1:10, the liposomes were clear and transparent with pale blue opalescence and a stable particle size, and the encapsulation efficiency was greater than 90%. No significant changes were observed in any characteristic following the addition of 11-DGA-3-O-Gal to the liposomes. The appearance, morphology, size distribution, and zeta potential distribution are presented in Figure 3.

In Vitro Release of Cantharidin (CTD)
The in vitro release of CTD from CTD-lip and 11-DGA-3-O-Gal-CTD-lip was studied, and this experiment was carried out in phosphate-buffered saline (PBS, pH = 7.4) with 0.25% Tween-80. As shown in Figure 4, after encapsulation of CTD into liposomes, the two nano-formulations showed a

In Vitro Release of Cantharidin (CTD)
The in vitro release of CTD from CTD-lip and 11-DGA-3-O-Gal-CTD-lip was studied, and this experiment was carried out in phosphate-buffered saline (PBS, pH = 7.4) with 0.25% Tween-80. As shown in Figure 4, after encapsulation of CTD into liposomes, the two nano-formulations showed a similar drug release profile, with~70% released in an initial burst followed by a slow phase. Both reached~90% of cumulative drug release after 24 h. The release curve of CTD in the liposomes conformed to the Weibull equation, as shown in Table 2. These results indicated that the addition of targeting molecules had no significant effect on the release of liposomes, which will be beneficial for the pharmacodynamics processes in the body. similar drug release profile, with ~70% released in an initial burst followed by a slow phase. Both reached ~90% of cumulative drug release after 24 h. The release curve of CTD in the liposomes conformed to the Weibull equation, as shown in Table 2. These results indicated that the addition of targeting molecules had no significant effect on the release of liposomes, which will be beneficial for the pharmacodynamics processes in the body.

In Vitro Cytotoxicity Assay
The cytotoxic effects of various liposomal formulations in HepG2 and L-02 cells are shown in Figure 5. The results indicated that both CTD-lip and modified-CTD-lip inhibited the proliferation of HepG2 and L-02 cells, and the inhibitory effect on L-02 cells was stronger than that on HepG2 cells. Under the same experimental conditions, the HepG2 cytotoxicity results indicated that the IC50 value of 11-DGA-3-O-Gal-CTD-lip was 0.772 ± 0.021 μg/mL, and compared with that of CTD-lip, the inhibitory effect on HepG2 cell proliferation was 1.64 times higher.

In Vitro Cytotoxicity Assay
The cytotoxic effects of various liposomal formulations in HepG2 and L-02 cells are shown in Figure 5. The results indicated that both CTD-lip and modified-CTD-lip inhibited the proliferation of HepG2 and L-02 cells, and the inhibitory effect on L-02 cells was stronger than that on HepG2 cells. Under the same experimental conditions, the HepG2 cytotoxicity results indicated that the IC 50 value of 11-DGA-3-O-Gal-CTD-lip was 0.772 ± 0.021 µg/mL, and compared with that of CTD-lip, the inhibitory effect on HepG2 cell proliferation was 1.64 times higher.
Molecules 2019, 24, x FOR PEER REVIEW 6 of 18 similar drug release profile, with ~70% released in an initial burst followed by a slow phase. Both reached ~90% of cumulative drug release after 24 h. The release curve of CTD in the liposomes conformed to the Weibull equation, as shown in Table 2. These results indicated that the addition of targeting molecules had no significant effect on the release of liposomes, which will be beneficial for the pharmacodynamics processes in the body.

In Vitro Cytotoxicity Assay
The cytotoxic effects of various liposomal formulations in HepG2 and L-02 cells are shown in Figure 5. The results indicated that both CTD-lip and modified-CTD-lip inhibited the proliferation of HepG2 and L-02 cells, and the inhibitory effect on L-02 cells was stronger than that on HepG2 cells. Under the same experimental conditions, the HepG2 cytotoxicity results indicated that the IC50 value of 11-DGA-3-O-Gal-CTD-lip was 0.772 ± 0.021 μg/mL, and compared with that of CTD-lip, the inhibitory effect on HepG2 cell proliferation was 1.64 times higher.

Inhibition Mechanism of 11-DGA-3-O-Gal-CTD-Lip in HepG2 Cells
A rapid proliferation rate is a prominent feature of tumor cells. Therefore, inhibition of the occurrence and development of tumor cells and promotion of tumor cell apoptosis are among the relevant factors to be considered for anti-tumor drugs. We investigated the effect of 11-DGA-3-O-Gal-CTD-lip on the cell cycle and apoptosis using flow cytometry. The results showed that the percentage of cells in G2 phase of the cell cycle significantly increased from (27.10 ± 1.99)% to (41.72 ± 1.79)% after treatment with 11-DGA-3-O-Gal-CTD-lip compared to 42.62 ± 1.09% after treatment with CTD-lip at the IC 50 concentration ( Figure 7A,B). The results were similar at the 1/2IC 50 concentration, suggesting that 11-DGA-3-O-Gal-CTD-lip induced the accumulation of HepG2 cells in S phase of the cell cycle. Moreover, the cell apoptosis results indicated that the cells treated with 11-DGA-3-O-Gal-CTD-lip had a lower apoptotic rate (31.36 ± 1.14)% than the cells treated with CTD-lip (50.68 ± 3.06)% at the IC 50 concentration ( Figure 7C,D). Compared with the blank control group, the proportion of early apoptosis and late apoptosis of cells treated with CTD liposomes increased significantly, and the difference in the apoptosis rate at each concentration group was statistically significant (p < 0.01). Comparison of the 11-DGA-3-O-Gal-CTD-lip group with the CTD-lip group revealed that the apoptosis rate of the liposomes did not significantly change after the modification with the targeting molecule, indicating that the effect of CTD liposomes on the apoptosis of HepG2 cells may be independent of the addition of the targeting molecule.

Inhibition Mechanism of 11-DGA-3-O-Gal-CTD-Lip in HepG2 Cells
A rapid proliferation rate is a prominent feature of tumor cells. Therefore, inhibition of the occurrence and development of tumor cells and promotion of tumor cell apoptosis are among the relevant factors to be considered for anti-tumor drugs. We investigated the effect of 11-DGA-3-O-Gal-CTD-lip on the cell cycle and apoptosis using flow cytometry. The results showed that the percentage of cells in G2 phase of the cell cycle significantly increased from (27.10 ± 1.99)% to (41.72 ± 1.79)% after treatment with 11-DGA-3-O-Gal-CTD-lip compared to 42.62 ± 1.09% after treatment with CTD-lip at the IC50 concentration ( Figure 7A,B). The results were similar at the 1/2IC50 concentration, suggesting that 11-DGA-3-O-Gal-CTD-lip induced the accumulation of HepG2 cells in S phase of the cell cycle. Moreover, the cell apoptosis results indicated that the cells treated with 11-DGA-3-O-Gal-CTD-lip had a lower apoptotic rate (31.36 ± 1.14)% than the cells treated with CTDlip (50.68 ± 3.06)% at the IC50 concentration ( Figure 7C,D). Compared with the blank control group, the proportion of early apoptosis and late apoptosis of cells treated with CTD liposomes increased significantly, and the difference in the apoptosis rate at each concentration group was statistically significant (p < 0.01). Comparison of the 11-DGA-3-O-Gal-CTD-lip group with the CTD-lip group revealed that the apoptosis rate of the liposomes did not significantly change after the modification with the targeting molecule, indicating that the effect of CTD liposomes on the apoptosis of HepG2 cells may be independent of the addition of the targeting molecule.

In Vivo Pharmacokinetics Study
The pharmacokinetic properties of 11-DGA-3-O-Gal-CTD-lip and CTD-lip were studied by detecting the CTD content in rat plasma. The mean plasma concentration-time curves of CTD after intravenous administration of 11-DGA-3-O-Gal-CTD-lip and CTD-lip are shown in Figure 8. The drug-time curve trends of the two liposomes were similar, and the drug was eliminated faster in the first 60 min, which may be related to the rapid elimination of free drugs from the

In Vivo Pharmacokinetics Study
The pharmacokinetic properties of 11-DGA-3-O-Gal-CTD-lip and CTD-lip were studied by detecting the CTD content in rat plasma. The mean plasma concentration-time curves of CTD after intravenous administration of 11-DGA-3-O-Gal-CTD-lip and CTD-lip are shown in Figure 8. The drug-time curve trends of the two liposomes were similar, and the drug was eliminated faster in the first 60 min, which may be related to the rapid elimination of free drugs from the The drug-time curve trends of the two liposomes were similar, and the drug was eliminated faster in the first 60 min, which may be related to the rapid elimination of free drugs from the liposome preparation and the liposome burst effect. After 60 min, the elimination of the drug in the blood slowed down. A non-compartment model was suitable to evaluate the plasma drug concentration time curves obtained in rats based on the analysis of models and parameters. The main pharmacokinetic parameters are summarized in Table 3. Compared with that of CTD-lip, the elimination half-life (T 1/2β ) of 11-DGA-3-O-Gal-CTD-lip decreased, the value of which was 2.42 ± 1.03 h. The results indicated that 11-DGA-3-O-Gal-CTD-lip could rapidly distribute to tissues from the blood. The elimination rate of 11-DGA-3-O-Gal-CTD-lip was faster than that of CTD-lip, indicating that 11-DGA-3-O-Gal-CTD-lip might be recognized quickly by the ASGPR or GA receptor. Meanwhile, the mean plasma clearance (CL) of 11-DGA-3-O-Gal-CTD-lip (0.74 ± 0.13 L/h·kg) was higher than that of CTD-lip (0.57 ± 0.10 L/h·kg). Moreover, the mean residence time (MRT) of 11-DGA-3-O-Gal-CTD-lip (2.47 ± 1.21 h) was shorter than that of CTD-lip (5.14 ± 1.16 h). These results indicated that 11-DGA-3-O-Gal-CTD-lip was eliminated more rapidly than CTD-lip from the circulation system. In addition, the central chamber distribution volume (Vc) results for 11-DGA-3-O-Gal-CTD-lip (0.52 ± 0.15 L/kg) and CTD-lip (0.46 ± 0.19 L/kg) suggested that the modified liposomes easily distributed into tissues, which will aid in improving the therapeutic target effect. The area under the curve of drug concentration (AUC 0-∞ ) of 11-DGA-3-O-Gal-CTD-lip was about 1.14 times less than that of CTD-lip (756.38 ± 15.12 µg/L·h). The AUC of 11-DGA-3-O-Gal-CTD-lip decreased as the value of CL increased, which indicated that the modification of liposomes were associated with the rapid removal of CTD from plasma. These results demonstrated that the modification of liposomes with 11-DGA-3-O-Gal had significant effects on the pharmacokinetics compared with those of CTD-lip. Table 3. Pharmacokinetic parameters in rat plasma after intravenous administration of CTD-lip and 11-DGA-3-O-Gal-CTD-lip (mean ± SD, n = 6).

Tissue Distribution Study
The concentrations of CTD in the heart, liver, spleen, lung, and kidney were determined at various time points after intravenous administration of CTD-lip and 11-DGA-3-O-Gal-CTD-lip. The concentration-time profiles in the tissues are shown in Figure 9. These results indicate the distribution trends of the different CTD formulations in vivo for rats. We found that the liver concentration of 11-DGA-3-O-Gal-CTD-lip was significantly higher than that of CTD-lip. This result indicated that the liposomes modified with 11-DGA-3-O-Gal could deliver the drug rapidly to the liver after intravenous administration and supported our assumption that liposomes modified with 11-DGA-3-O-Gal can enhance liver-targeting through the receptor. Furthermore, the concentration-time data were quantitively analyzed to define the livertargeting ability. The pharmacokinetic parameters AUC0-t and Cmax in various tissues were determined, and the results are summarized in Table 4. Then, the important parameters for the evaluation of targeting ability, including targeting efficiency (Te), relative targeting efficiency (RTe), relative uptake rate (Re), and peak concentration ratio (Ce), were calculated using AUC0-t and Cmax. The data are listed in Tables 4 and 5. Te indicates the selectivity of a CTD formulation to target tissue. The Te of CTD-lip in the liver and kidney was (26.93 ± 2.65)% and (29.98 ± 2.43)%, respectively, demonstrating that the highest selectivity rate was in the kidney. Table 4. Pharmacokinetic parameters of CTD-lip and 11-DGA-3-O-Gal-CTD-lip in rat tissues (mean ± SD, n = 6).  Furthermore, the concentration-time data were quantitively analyzed to define the liver-targeting ability. The pharmacokinetic parameters AUC 0-t and C max in various tissues were determined, and the results are summarized in Table 4. Then, the important parameters for the evaluation of targeting ability, including targeting efficiency (T e ), relative targeting efficiency (R Te ), relative uptake rate (R e ), and peak concentration ratio (C e ), were calculated using AUC 0-t and C max . The data are listed in Tables 4 and 5. T e indicates the selectivity of a CTD formulation to target tissue. The T e of CTD-lip in the liver and kidney was (26.93 ± 2.65)% and (29.98 ± 2.43)%, respectively, demonstrating that the highest selectivity rate was in the kidney. Table 4. Pharmacokinetic parameters of CTD-lip and 11-DGA-3-O-Gal-CTD-lip in rat tissues (mean ± SD, n = 6).  Table 5. Targeting parameters of CTD-lip and 11-DGA-3-O-Gal-CTD-lip in rat tissues (mean ± SD, n = 6). Compared with that of CTD-lip, the T e of 11-DGA-3-O-Gal-CTD-lip in liver reached (41.15 ± 3.28)%, indicating that 11-DGA-3-O-Gal was specifically recognized by receptors. Strictly speaking, the relative targeting efficiency (R Te ) should be a comparison between the targeted preparation and the non-targeted preparation. Because cantharidin is insoluble in water, it is difficult to establish a non-targeted preparation group (cantharidin solution group), so R Te is here defined as the comparison of CTD-lip and modified liposomes. The results showed that 11-DGA-3-O-Gal-CTD-lip increased the liver targeting by 1.53 times compared to CTD-lip, indicating significant liver targeting. R e indicates the targeting ability of a liposomal formulation. An R e greater than 1 indicates that the modified liposomes have a greater live-targeting ability than CTD-lip. The R e of 11-DGA-3-O-Gal-CTD-lip was 1.69 times higher than that of CTD-lip. C e indicates the effect of a liposome formulation on drug distribution. The results for the C e of 11-DGA-3-O-Gal-CTD-lip were in accordance with the T e and R e results, demonstrating that 11-DGA-3-O-Gal-CTD-lip was more optimally recognized by the liver. Therefore, compared with CTD-lip, the addition of modified molecule improved the selectivity of the liposomes to the liver and exhibited active targeting to the liver.

Synthesis of 11-DGA-3-O-Gal
11-DGA-3-O-Gal was synthesized using DGA(I) as a hydrophobic segment and Gal as a hydrophilic segment. DGA (Scheme 1I) was synthesized by Clemmensen reaction. Zn-Hg (0.45 g) in dioxane were added to the GA (0.72 g) solution. Then 1.5 mL HCl (12 mol/L) were slowly added within 30 min, the mixture was stirred at 10 • C for 3 h. TLC was used for identification in a silica gel G plate with ethyl acetate-petroleum ether (1:5) as a developing solvent. The obtained solution was placed in a beaker, and distilled water was added to induce precipitation. The obtained precipitate was collected by filtration and dried at 40 • C. The crude products were purified by silica gel column chromatography with ethyl acetate-petroleum ether (1:5) as a eluent. The yield of DGA was 86.32% and the purity was greater than 98%. until the wash solution was neutral, and dried at 40 °C. The chemical structure of 11-DGA-3-O-Gal(Scheme 1III) was confirmed by 13 C-NMR and 1 H-NMR (in CDCl3, 600 MHz).

Preparation of Liposomes
Liposomes were prepared by the classic method of thin-film evaporation. Briefly, to prepare the modified liposomes, soybean phospholipids (SPC), cholesterol, and CTD were dissolved in 15 mL chloroform at a mass ratio of 10:1:1. The chloroform was then removed using a rotary evaporator to form a dry-lipid film at 55 °C under uniform speed. The lipid film was hydrated with phosphatebuffered saline (PBS, pH 6.4) under magnetic stirring at 55 °C. Then, the resulting suspension was sonicated with a probe sonicator for 30 min with a 2 s interval, which produced small unilamellar liposomal vesicles. Finally, the obtained liposome solution was extruded to pass through 0.22 μm pore size of microporous membranes to prepare the liposomes with an uniform size.
The targeted liposomes 11-DGA-3-O-Gal-CTD-lip were prepared by the post-insertion method. SPC, cholesterol, CTD, and 11-DGA-3-O-Gal were dissolved in 15 mL chloroform at a mass ratio of 10:1:1:1, and the rest operations were the same as above.

Physicochemical Characterization of Liposomes
The physicochemical characterization of liposomes was performed to determine the morphology, particle size, zeta potential (ZP), polydispersity index (PDI), and encapsulation efficiency (EE). The surface morphologies of CTD-lip and 11-DGA-3-O-Gal-CTD-lip were analyzed using a transmission electron microscope (TEM). The particle size, ZP, and PDI of liposomes were determined using a Zetasizer Nano ZS90 analyzer (Malvern Instruments, UK). The liposomes were diluted with distilled water at room temperature before measurement.
Encapsulation efficiency (EE%) refers the ratio of drug-encapsulated in liposome (Wliposome) to the total amount of drug (Wtotal) in the liposome preparation. The EE was determined using gel exclusion chromatography. To calculate the Wliposome, about 0.5 mL of a liposome sample was added drop-wise to the top of the column and then passed through Sephadex G-50 and eluted with distilled water to separate the non-entrapped drug. The eluate containing the entrapped CTD was concentrated to 1 mL and then disrupted with a 9 mL methanol-acetonitrile mixture (at a ratio of 1:1, v/v) to calculate the Wliposome. Similarly, to determine the Wtotal in a liposome sample, a 4 mL methanolacetonitrile mixture (at a ratio of 1:1, v/v) was added to a 0.5 mL liposome suspension. After separation of the free drug from the liposomal formulation, the concentrations of Wliposome and Wtotal were measured by HPLC under the following conditions: BETASIL column: C18 (4.6 mm × 150 mm,

Preparation of Liposomes
Liposomes were prepared by the classic method of thin-film evaporation. Briefly, to prepare the modified liposomes, soybean phospholipids (SPC), cholesterol, and CTD were dissolved in 15 mL chloroform at a mass ratio of 10:1:1. The chloroform was then removed using a rotary evaporator to form a dry-lipid film at 55 • C under uniform speed. The lipid film was hydrated with phosphate-buffered saline (PBS, pH 6.4) under magnetic stirring at 55 • C. Then, the resulting suspension was sonicated with a probe sonicator for 30 min with a 2 s interval, which produced small unilamellar liposomal vesicles. Finally, the obtained liposome solution was extruded to pass through 0.22 µm pore size of microporous membranes to prepare the liposomes with an uniform size.
The targeted liposomes 11-DGA-3-O-Gal-CTD-lip were prepared by the post-insertion method. SPC, cholesterol, CTD, and 11-DGA-3-O-Gal were dissolved in 15 mL chloroform at a mass ratio of 10:1:1:1, and the rest operations were the same as above.

Physicochemical Characterization of Liposomes
The physicochemical characterization of liposomes was performed to determine the morphology, particle size, zeta potential (ZP), polydispersity index (PDI), and encapsulation efficiency (EE). The surface morphologies of CTD-lip and 11-DGA-3-O-Gal-CTD-lip were analyzed using a transmission electron microscope (TEM). The particle size, ZP, and PDI of liposomes were determined using a Zetasizer Nano ZS90 analyzer (Malvern Instruments, UK). The liposomes were diluted with distilled water at room temperature before measurement.
Encapsulation efficiency (EE%) refers the ratio of drug-encapsulated in liposome (W liposome ) to the total amount of drug (W total ) in the liposome preparation. The EE was determined using gel exclusion chromatography. To calculate the W liposome , about 0.5 mL of a liposome sample was added drop-wise to the top of the column and then passed through Sephadex G-50 and eluted with distilled water to separate the non-entrapped drug. The eluate containing the entrapped CTD was concentrated to 1 mL and then disrupted with a 9 mL methanol-acetonitrile mixture (at a ratio of 1:1, v/v) to calculate the W liposome . Similarly, to determine the W total in a liposome sample, a 4 mL methanol-acetonitrile mixture (at a ratio of 1:1, v/v) was added to a 0.5 mL liposome suspension. After separation of the free drug from the liposomal formulation, the concentrations of W liposome and W total were measured by HPLC under the following conditions: BETASIL column: C18 (4.6 mm × 150 mm, 5 µm), mobile phase: acetonitrile-water (40:60, v/v), flow rate: 1.0 mL/min, wavelength: 230 nm, and injection volume: 10 µL.
where W liposome is the weight of drug being encapsulated in the liposomes, W total is the weight of the total amount of charged drug.

In Vitro Release of CTD
The release of CTD from CTD-lip and 11-DGA-3-O-Gal-CTD-lip was determined at 37 • C using the dialysis bag method. First, a 1 mL liposome suspension was sealed in a dialysis bag. The bags were then placed in 300 mL of PBS buffer (pH 7.4) containing 0%, 0.25%, 0.5%, and 1% Tween-80 under magnetic stirring (360 r/min, 37 ± 0.5 • C). The release medium (1 mL) was sampled at pre-determined time intervals (5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 7 h, 24 h) and immediately replaced with the same volume of fresh medium. Then, 0.2 mL of octadecane methanol solution (7.5 µg/mL) as an internal standard was added to the samples, which were then passed through a microporous membrane filter with a 0.22 µm pore size. The concentration of CTD was measured using gas chromatography-mass spectrometry (GC-MS). The release experiments were conducted in triplicate. The accumulated release values for CTD-lip and 11-DGA-3-O-Gal-CTD-lip were calculated using the following formula: Drug release percentage (%) = (W t /W total ) × 100% (2) where W t represents CTD release at different time point, and W total is the amount of drug in the liposomes.
MTT colorimetric assays were used to evaluate the effect of liposomes on the survival rate of HepG2 and L-02 cells. Cells (7000 cells/well) were seeded in 96-well plates and incubated for 16 h at 37 • C. Then, the culture medium was replaced with 100 µL of medium containing CTD-lip or 11-DGA-3-O-Gal-CTD-lip at different CTD concentrations (0.05, 0.15, 0.25, 0.5, 1, 2, 3, 4, 5 µg/mL). After treatment for 24 h, 48 h, 72 h and 96 h, the cells were washed with PBS twice, and then, 100 µL MTT (0.5 mg/mL) was added to each well followed by incubation for another 4 h in the dark at 37 • C. The medium was then discarded, and 150 µL dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals by shaking in a horizontal oscillator for 15 min. The absorbance at 492 nm was measured with an automatic enzyme standard instrument. The inhibition rate (IR) of cellular proliferation was calculated as follows: The half-maximal inhibitory concentration (IC 50 ) values were calculated using SPSS.

Cell Migration Assays
The effects of CTD-lip and 11-DGA-3-O-Gal-CTD-lip on cell migration were assessed by the transwell chamber method. A transwell chamber was placed in a 24-well plate, and DMEM high-glucose medium containing 10% FBS was added to the lower chamber of the transwell. HepG2 cells in logarithmic growth phase were digested into a single cell suspension using trypsin. After washing twice in serum-free medium, the cells were counted and adjusted to a cell concentration of 1 × 10 4 cells/mL, and 100 µL of the cell suspension was added to the inner chamber followed by incubation for 1~1.5 h. Then, aliquots of 100 µL of different concentrations of liposome solution were added to the inner chamber, and incubation was continued for 24 h. After the culture medium was discarded, the cells were washed three times with PBS and fixed with 4% paraformaldehyde solution for 20 min. The cell nuclei were stained with crystal violet (0.5%) for 30 min. The cells were then rinsed three times with PBS. Images of five random fields were taken under a microscope. The effect of liposomes on cell migration was evaluated by calculating the migration rate as follows: Migration rate = Cell number in experimental group/Cell number in control group × 100% (4)

Cell-Cycle and Cell Apoptosis Detection
HepG2 cells were treated with different liposomes for 48 h. For all treatments, the respective drug concentrations were maintained at IC 50 and 1/2IC 50 . After 48 h of incubation, the cell culture was aspirated into a suitable centrifuge tube, and cells were washed twice with PBS and then digested with 0.25% trypsin. After centrifugation at 1000 r/min for 5 min, the supernatant was discarded and the substratum was resuspended in PBS and adjusted to a cell concentration of 1 × 10 5 cells/mL. Following the manufacturer's protocol, the cell cycle and cell apoptosis was assessed using flow cytometry.

Pharmacokinetic Studies
Sprague Dawley(SD) rats (220 ± 20 g, male and female rats in equal numbers) were fed a standard laboratory diet with free access to water at a controlled temperature of 25 ± 2 • C and relative humidity of 60 ± 5% with a 12 h light/dark cycle. Animals were fed adaptively for 3d and kept fasting for 12 h with free access to water before experiments. The pharmacokinetic properties of CTD-lip and 11-DGA-3-O-Gal-CTD-lip were evaluated via determination of the CTD content in rat plasma. Eighteen experimental rats were randomly divided into three groups, the CTD-lip, 11-DGA-3-O-Gal-CTD-lip, and saline groups. Each group of rats was injected with a dose of 3 mg/kg via the tail vein. After injection, blood samples (0.3 mL) were collected from the retroorbital plexus at different time point (5,15,30,45, 60, 90, 120, 150 min) and immediately centrifuged (4000 rpm, 20 min). Then, 100 µL of the upper plasma sample was added to 300 µL methanol followed by mixing for 3 min by vortex. After centrifugation (4000 rpm, 20 min), the supernatant was transferred to a centrifuge tube, and 50 µL octadecane (0.5 µg/mL) was added as an internal standard. The mixed solution was dried by nitrogen. The residue was reconstituted with 200 µL of methanol, vortex-mixed for 3 min, and then centrifuged at 12,000 r/min for 20 min (4 • C). Subsequently, the supernatant was examined using GC-MS. Pharmacokinetic data were analyzed with DPSv17.10.

Tissue Distributions
SD rats (220 ± 20 g, male and female rats in equal numbers) were fed a standard laboratory diet with free access to water at a controlled temperature of 25 ± 2 • C and relative humidity of 60 ± 5% with a 12 h light/dark cycle. Animals were fed adaptively 3 days and kept fasting for 12 h with free access to water before experiments. One hundred forty-four experimental rats were randomly divided into three groups: the CTD-lip, 11-DGA-3-O-Gal-CTD-lip, and saline groups. Each group of rats was injected with a dose of 3 mg/kg via the tail vein. After injection, rats were euthanized immediately at different time point (5,15,30,45, 60, 90, 120, 150 min), and the hearts, livers, spleens, lungs, and kidneys were collected. Tissue samples were washed with normal saline, wiped with filter paper, weighed, and homogenized in equal volumes of normal saline (w/v). Then, 500 µL of homogenate was added to 1500 µL methanol followed by mixing for 3 min by vortex. After centrifugation (4000 rpm, 20 min), 3 mL of the supernatant was transferred to a centrifuge tube, and 250 µL octadecane (0.5 µg/mL) was added as an internal standard. These steps were performed according to the standard methods for plasma preparation in pharmaceutics. Finally, the supernatant was detected using GC-MS. The parameters were measured using a non-compartmental analysis with DPSv17.10. Based on the AUC and C max data, the targeting parameters of each CTD liposome in a tissue were calculated, including T e , R Te , R e and C e [41]. The parameters were calculated as follows: Te(%) = (AUC target /AUC total ) × 100% R Te = Te modified liposome /Te CTD-lip (6) R e = (AUC modified liposome /AUC CTD-lip ) × 100% (7) C e = [(C max ) modified liposome /(C max ) CTD-lip ] × 100% (8)

Statistical Analysis
All data were generated in triplicate and expressed as the mean ± standard deviation (SD). Statistical comparisons between two groups were performed using Student's t-test, and multiple comparisons were performed using one-way analysis of variance (ANOVA). All statistical analyses were performed using SPSS 21.0. Results were considered to statistically significant at p < 0.05.

Conclusions
As previously described, cantharidin has a positive therapeutic effect on liver tumors, but its application has been limited by its water insolubility, low bioavailability, and high toxicity. In order to overcome these barriers of cantharidin in clinical application, we developed and characterized a novel, lipid-based nanocarrier for CTD, which can target hepatoma cells without the limitations of CTD.
11-DGA-3-O-Gal was synthesized from DGA and bromine acetyl galactose glucoside. The chemical structure of 11-DGA-3-O-Gal was confirmed by NMR. In addition, 11-DGA-3-O-Gal was successfully incorporated into liposomes containing CTD. 11-DGA-3-O-Gal-CTD-lip had a particle size of less than 110 nm, with an EE lager than 90% and a sustained release for 24 h in vitro. To assess the new delivery system of 11-DGA-3-O-Gal-CTD-lip, we investigated the anti-cancer activities of CTD-lip and 11-DGA-3-O-Gal-CTD-lip in vitro and in vivo.
In vitro, compared to CTD-lip, 11-DGA-3-O-Gal-CTD-lip displayed higher cytotoxicity and migration inhibition on HepG2 cells, but did not increase the apoptotic rate of cells. The pharmacokinetic study in rats showed that 11-DGA-3-O-Gal-CTD-lip was eliminated more rapidly than CTD-lip. These results suggested that the CTD liposomes modified with 11-DGA-3-O-Gal are an efficient target carrier for the treatment of hepatocellular carcinoma. Furthermore, the tissue distribution of 11-DGA-3-O-Gal-CTD-lip was investigated, and we found that the T e , R Te , R e and C e of CTD in the liver were higher than in other tissues, demonstrating that 11-DGA-3-O-Gal-CTD-lip had an excellent effect of liver targeting. These results supported our hypothesis that liposomes containing 11-DGA-3-O-Gal ligand, is a potential drug delivery carrier for hepatic diseases.