Enhanced hepatic-targeted delivery via oral administration using nanoliposomes functionalized with a novel DSPE–PEG–cholic acid conjugate

Abstract

Since cholic acid receptors are rich on the membrane of intestine epithelial cells and hepatocytes, cholic acid was conjugated to DSPE–PEG, and functional nanoliposomes loaded with model drug silybin (CA–LPs–silybin) were constructed and characterized for oral administration and to target the liver. CA–LPs–silybin was found stable in terms of nanoliposome integrity after cellular transport via TEM imaging and HPLC analysis of the % of encapsulated silybin. The investigation of CA–LPs–silybin includes its transport and mechanisms across the ASBT-positive Caco-2 cell monolayers, its hepatic targeting efficiency and mechanisms via in vitro cell uptake studies in the NTCP-positive HepG2 cells and in vivo hepatic distribution. The results of the investigation showed that CA–LPs–silybin exhibited increased levels of intracellular transport and uptake in vitro and liver accumulation in vivo as compared with unmodified nanoliposomes, via a cholic acid receptor mediated mechanism. Inhibitor competition experiments suggest that CA–LPs–silybin is transported across Caco-2 cell monolayers through a transcellular but not a paracellular pathway and enters HepG2 cells through an unspecific endocytosis pathway and a specific lipid raft and clathrin-mediated mechanism.

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1 Introduction

Liver diseases are the major causes of disability and mortality worldwide. Chemotherapy has been limited for its poor specificity. Many potent drugs are often not effective enough in vivo or exhibit adverse effects, thus drug targeting to the liver may represent a promising strategy.¹

Hepatic-targeting drug delivery by oral administration has attracted much attention in research of low bioavailability or toxic drugs because of the advantages of enhanced distribution in target sites and reduced side effects to other tissues. Active targeting strategies rely on the biomarkers that are specifically overexpressed on target organ. These biomarker molecules function as specific receptors on cell membrane for recognizing and binding extracellular specific ligands. Ligand binding activates the receptor and subsequently induces the cellular uptake of nano-medicines via endocytosis pathway.²

Nanoliposomes are frequently reported as the preferred drug delivery systems because of its biocompatibility and biodegradability³ via different fabrication methods, nanoliposomes can be obtained with desired particle size from 50 nm to 300 nm. Drugs with different solubility characteristics, water-soluble or water insoluble, can all be loaded into nanoliposomes by currently available techniques. However nanoliposomes had poor stability in vivo and liver targeting specificity via oral administration, because it may distributed in liver, spleen, lung and bone marrow that was rich in reticuloendothelial system by recognition and phagocytosis as a foreign body.^4,5

Many efforts have been dedicated to the use of poly(ethylene glycol) that are preferred to increase its hydrophilicity and to reduce gastrointestinal enzymatic degradation and the uptake by phagocytes in blood circulation on the surface of nanoliposomes,^6–9 but the poor liver targeting specificity of nanoliposomes was still not solved.

Another approach for hepatic-targeting delivery of drugs via oral administration has been dedicated to the use of cholic acid receptor on liver.^10,11 Targeting via cholic acid modified carriers exploits highly specific interaction of cholic acid ligand with ASBT receptors which is specifically and abundantly present on intestinal epithelium cells^12,13 and NTCP receptors which is specifically and abundantly present on hepatocytes.^14,15 By coupling cholic acid (CA) moieties to the nanoliposomes, the uptake of drug-loaded systems into hepatocytes would be enhanced with high degree of selectivity. The in vivo fate of cholic acid is demonstrated in ESI Fig. S1.†^16–19 However, most researchers utilized cholic acid receptor by the development of cholic acid prodrugs,^20,21 prodrugs altered the characteristics of drugs, which may influence pharmacokinetic and pharmacodynamic behaviors of drugs.

Based on above background, it is supposed that the conjugation of cholic acid on the surface of nanoliposomes may lead to their favorable uptake and transport in GI endothelial cells and hepatic cells providing a potential delivery system for orally hepatic targeting drug delivery. For the proof-of-concept, we described the construction of cholic acid modified nanoliposomes (CA–LPs) bearing cholic acid moieties on the surface. Silybin, as a model drug for this study, is a hepatoprotectant and has widely been used in treatment of various liver disorders, such as cirrhosis, hepatitis, and fatty infiltration due to alcohol and toxins in Chinese traditional medicine. In order to study the trans-cellular process of CA–LPs and determine the mechanism among endocytosis, exocytosis and transcytosis, a human colon carcinoma cell line, Caco-2 that overexpressed ASBT, is chosen as the model for intestine epithelial barrier.^22–24 Cellular uptake of CA–LPs was also evaluated in HepG2 cells, a human liver carcinoma cell line that overexpressed NTCP.²⁵ The transport of the functional nanoliposomes loaded with silybin (CA–LPs–silybin) in Caco-2 were studied using unmodified liposmes (LPs–silybin) as the control, followed by in vitro HepG2 uptake and in vivo hepatic distribution studies. The mechanisms involved in these process were explored as well.^26,27

2 Experimental

2.1 Materials

N-Hydroxysuccinimidyl–PEG2000–DSPE and DSPE–PEG2000 were purchased from NOF Corporation (Tokyo, Japan). Soybean phosphatidylcholine (SPC) was obtained from LIPOID (Germany). Cholesterol was supplied from Sigma-Aldrich (St. Louis, MO, USA). Cholic acid was supplied from Sigma-Aldrich (St. Louis, MO, USA). Sephdax LH-20 was from GE Healthcare (Fairfield, CT, USA). Anhydrous N,N-dimethylformamide (DMF), hydroxybenzotriazole (HOBT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), chlorpromazine, filipin, methyl-beta-cyclodextrin (MβCD), nystatin and genistein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silybin was kindly provided by Sino-herb bio-technology Co., Ltd (Xi'an, China),DIO (3,3′-dioctadecyloxacarbocyanine perchlorate) was supplied from Beijing Fanbo Biochemicals CO. LTD. (Beijing, China).

The human colon carcinoma cell line Caco-2 and human liver carcinoma cell line HepG2 were obtained from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Caco-2 cells were cultured in MEM medium containing a final concentration of 10% (v/v) fetal bovine serum (FBS), 1% nonessential amino acid and 1% antibiotics (penicillin, 100 U mL⁻¹ plus streptomycin). HepG2 cells were cultured in RPMI 1640 medium containing a final concentration of 10% (v/v) fetal bovine serum (FBS), 1% nonessential amino acid and 1% antibiotics (penicillin, 100 U mL⁻¹ plus streptomycin). Both cell lines were cultured at 37 °C in humidified atmosphere with 5% CO₂.^28–30

The male ICR mice of 18–20 g were obtained from the Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC) Health Science Center (Beijing, China), and kept under SPF condition for 1 week before the study, with free access to standard food and water. All studies in mice were performed in accordance with guidelines approved by the Ethics Committee of the Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC).

2.2 Synthesis of DSPE–PEG–cholic acid

In brief, DSPE–PEGNH₂ and cholic acid (1 : 1; molar ratio) in DMF were reacted for 48 h. The conjugation efficiency was monitored by thin-layer chromatography (TLC). After dialysis and freeze-drying, the targeting material was confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF; Bruker Daltonics, USA).

2.3 Preparation and characterization of nanoliposomes

The silybin-loaded nanoliposomes modified with cholic acid (CA–LPs–silybin) or unmodified (LPs–silybin) were prepared by ethanol injection method. The particle sizes, polydispersity indexes (PDI) and zeta-potential values of all nanoliposomes were measured using Nano Series ZS Zeta sizer (Malvern Instruments Ltd., U.K). The size and morphology of silybin encapsulated nanoliposomes were examined using transmission electron microscopy (TEM, Tecnai G2 20ST, FEI Co., Japan). Silybin was quantitated with high performance liquid chromatography (HPLC) system with a UV detector (Shimadzu, JAPAN). A C18 column (Kromasil, 250 × 4.6 mm, 5 μm) was used for the analysis with column temperature of 30 °C. The mobile phase was consisted of methanol and water (48/52, v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was set at 287 nm. All samples were analyzed in triplicate. To estimate the encapsulation efficiency of silybin nanoliposomes, the nanoliposomes suspensions were destroyed by adding methanol. The solution was properly diluted prior to HPLC analysis. Encapsulation ratio and loading efficiency were calculated with the following formulas:

Encapsulation ratio (%) = (weight of silybin in found loaded/weight of silybin input) × 100 (1)

Loading efficiency (%) = (weight of silybin found loaded/weight of drug-loaded nanoliposomes) × 100 (2)

2.4 Receptor expression study

Caco-2 and HepG2 cells were seeded in a 6-well plate at a density of 1 × 10⁶ per well for 24 hours prior to study. The medium was removed and cells were washed with PBS. Cell monolayers were first incubated with 1 mg mL⁻¹ cholic acid for 30 min or 1 h at 37 °C, then washed with PBS, trypsinization, PBS again followed by two cycles of freezing and thawing and centrifugation for 20 min. The supernatant was collected for Elisa (bio-function technology Co. Ltd., Beijing, China) and BCA (cw0014, CWBIO, China) analysis.^31,32

2.5 Demonstration of the transport of nanoliposomes across the Caco-2 cell monolayer

2.5.1 Transport of nanoliposomes across the cell monolayer. To investigate the influence of different silybin formulations on adsorption properties of silybin, Caco-2 cells were utilized as an in vitro model of gastrointestinal epithelium. Caco-2 cells were harvested at 80% confluence with 0.25% trypsin, and seeded onto polycarbonate membrane filters (0.4 mm pore size, 1.12 cm² growth area) in Transwell cell culture chambers (Corning Costar, Cambridge, MA) at a density of 2 × 10⁵ cells per insert. The culture medium (0.5 mL per insert and 1.5 mL per well) was replaced everyday. After 21 days culture, cell monolayers were used for the following assays. Before experiments, cell monolayers were washed twice with D-Hank's buffered salt solution (Ca²⁺ and Mg²⁺ free, 137.93 mM NaCl, 5.33 mM KCl, 4.17 mM NaHCO₃, 0.44 mM KH₂PO₄, 0.34 mM Na₂HPO₄, 5.56 mM D-glucose, HBSS) at 37 °C for 30 min. The transepithelial electrical resistance (TEER) of monolayer was measured using a Millicell-ER system (Millipore Corporation, Bedford, MA) to determine the formation of the monolayer and its integrity during the experiment. Only the cell monolayers with TEER values over 800 Ω cm² were used. To evaluate the transports, various formulations of silybin were diluted with D-HBSS to a final concentration of 50 μg mL⁻¹ silybin as the test solutions, including free silybin, LPs–silybin and CA–LPs–silybin. The drugs were applied to Caco-2 cell monolayers from the apical to basolateral direction by adding 0.5 mL of a test solution at the apical side and adding 1.5 mL of D-HBSS at the basolateral side. A volume of 1.5 mL sample was taken from the basolateral side at 2 h. 500 μL of samples were mixed with 500 μL methanol and shaken for 1 min using a vortex mixer, and centrifuged at 8000 rpm for 10 min. The supernatant was injected into the HPLC system for measuring silybin content. Apparent permeability coefficient (Papp) was calculated with the following equation: Papp = dQ/dt × 1/(AC₀), where dQ/dt is the permeability rate, C₀ is the initial concentration at the apical side and A is the surface area of a monolayer.³³

2.5.2 The direct imaging of nanoliposomes in apical and basolateral chamber by TEM. To directly observe the integrity of the CA–LPs–silybin after transporting across the cell monolayer, cell monolayers were incubated with D-HBSS (control) or with LPs–silybin and CA–LPs–silybin at 37 °C for 2 h. The basolateral medium was collected, treated with phosphotungstic acid, a contrasting agent preferentially fixes the periphery of the particles, and observed under a transmission electron microscopy (TEM). The particle sizes in the basolateral medium was measured using Nano Series ZS Zeta sizer.³⁴

2.6 Mechanistic studies on the transport of CA–LPs–silybin³⁵

2.6.1 Pathway studies with endocytosis inhibitors. In order to identify the transcellular transport mechanism of CA–LPs–silybin, transport experiments were performed in the presence of specific inhibitors with different types of endocytosis. Cell monolayers were first incubated with 50 μg mL⁻¹ nystatin, 50 μg mL⁻¹ genistein, 10 μg mL⁻¹ chlorpromazine, 1 mg mL⁻¹ NaN₃, or 10 mM methyl-b-cyclodextrin (MβCD) at 37 °C for 30 min. After the aspiration of pre-incubation solutions, washed with PBS buffer and 50 μg mL⁻¹ CA–LPs–silybin was added and further incubated at 37 °C for 2 h. To inhibit ASBT receptor, cell monolayers were first incubated with 1 mg mL⁻¹ cholic acid (Sigma, Beijing local agent, China) at 37 °C for 30 min. After the aspiration of cholic acid, washed with PBS buffer and 50 μg mL⁻¹ CA–LPs–silybin was added and further incubated at 37 °C for 2 h.

2.6.2 Effect of EGTA. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N'-tetraacetic acid tetra-sodium salt (EGTA) solution was prepared in D-HBSS (Ca²⁺ and Mg²⁺ free, 137 mM NaCl, 5.36 mM KCl, 0.44 mM KH₂PO₄, 0.34 mM Na₂HPO₄, 4.17 mM NaHCO₃, 5.6 mM glucose). Cell monolayer were treated with EGTA for 30 min at 37 °C. After the aspiration of EGTA, washed with PBS buffer and 50 μg mL⁻¹ CA–LPs–silybin was added and further incubated at 37 °C for 2 h. The same test with the same batch of CA–LPs–silybin without pre-incubation EGTA was served as the negative control. Finally, Papp was determined.

2.6.3 Effect of other factors. To demonstrate energy-dependent endocytosis, cell monolayers were first incubated at 4 °C, 22 °C or 37 °C (control) for 30 min before applying the CA–LPs–silybin. The cells were further incubated at 4 °C, 22 °C, or 37 °C (control) for 2 h after applying the CA–LPs–silybin.

2.7 Demonstration of NTCP mediated targeting delivery

2.7.1 In vitro cellular uptake. HepG2 cells were seeded in 6-well cell culture clusters (Corning, NY, USA) at a density of 1 × 10⁶ cells per well. On the following day, the confluent cells were incubated with silybin loated nanoliposomes including LPs–silybin and CA–LPs–silybin (containing silybin at 50 μg mL⁻¹) in serum-free medium at 37 °C for 2 h. Meanwhile, the cells treated with the medium alone were used as negative control. After incubation, the cells were washed 3 times with cold PBS, lysed, harvested and centrifuged to collect the supernatant. The mean concentration of silybin in cells was measured with HPLC (Shimadzu, JAPAN), the content of protein was measured with BCA Protein Quantitation Kit (cw0014, CWBIO, China) by microplate reader (Bio Tek, USA),³⁶ the intracellular drug concentration of the sample in each hole was standardized by dividing the total amount of protein expressed in each hole, in μg μg⁻¹ protein.

DIO was one kind of common fluorescence probe of cell membrane, it was choosen as fluorescence probe for confocal microscope studies, HepG2 cells were seeded in Petri dishes for 24 h, the medium was removed and cells were washed with PBS. After incubation with Dio at 37 °C for 2 h, the cells were washed three times with PBS and fixed with fresh 4% paraformaldehyde for 10 min at 22 °C. The cells were then counterstained with Hoechst 33258 for 10 min to stain the nucleic. Blue fluorescence of Hoechst 33258 and green fluorescence of Dio were observed using a Zeiss LSM780 CLSM (Zeiss Co., Germany).

2.7.2 Endocytosis pathway detection by uptake inhibitors. The HepG2 cells were pre-treated with different inhibitors for 30 min. The inhibitors and their concentrations were as follows: NaN₃, 1 mg mL⁻¹; genistein, 50 μg mL⁻¹; MβCD, 10 mM; nystatin, 50 μg mL⁻¹; chlorpromazine, 10 μg mL⁻¹; cholic acid, 1 mg mL⁻¹. Next, the inhibitors were removed, and the cells were incubated with modified nanoliposomes for 2 h. Then, the cellular uptake of nanoliposomes was measured as described in Section 2.7.1.

2.7.3 In vivo targeting efficiency.
2.7.3.1 Animals and dosing. Male ICR rats (body weight 20 ± 2 g) were fasted overnight. Each group consisted of six animals and received one of the formulations. The oral dose of silybin was 100 mg kg⁻¹ (nanoliposomes were freezen drying and dissolved with water to concentrate silybin concentration to 4 mg mL⁻¹). Blood samples were taken at time points of 0.25, 0.5, 1, 1.5, 2, and 4 h and the animals were immediately sacrificed and liver was isolated. The livers were washed with saline, blot dried and weighed.
2.7.3.2 Plasma processing and HPLC analysis. Plasma processing and HPLC analysis were performed according to literatures with a little modification.³⁷ An aliquot of 100 μL plasma and 1 mL aether was transferred to EP tubes and vortexed for 3 min, followed by the centrifugation at 12 000 rpm for 10 min. An aliquot of 20 μL supernatant was injected into the HPLC system and the silybin was detected. The standard curves were obtained, with a correlation coefficient of 0.9991 over the concentration range of 0.066–8.52 μg mL⁻¹. LOQ were 0.033 μg mL⁻¹. The within-day and between-day relative standard deviation did not exceed 5% for the same batch of reagents. The accuracy of the method was verified with recovery values of 80–120%.
2.7.3.3 Liver processing and HPLC analysis. The hepatic distribution of silybin was examined by the following method:³⁸ livers were individually homogenized with normal saline, 1 mL homogenization buffer was added to 3 mL aether. Subsequent steps were identical to those described above for the analysis of plasma samples. The standard curves were obtained, with a correlation coefficient of 0.9989 over the concentration range of 13.31–1704 ng g⁻¹. LOQ were 0.033 μg mL⁻¹. The within-day and between-day relative standard deviation did not exceed 5% for the same batch of reagents. The accuracy of the method was verified with recovery values of 80–120%.
2.7.3.4 Pharmacokinetic analysis. The pharmacokinetics parameters MRT (the mean retention time), and AUC_0–t (area under curve) were calculated from the drug concentration–time data according to noncompartmental methods by using kinetica software version 4.4 (Thermo Electron, USA). T_max was obtained from the time point corresponding to peak concentration of silybin. C_max was determined from peak concentration of silybin after intragastrical administration of silybin (100 mg kg⁻¹). The hepatic targeting parameters were calculated with the formulas:³⁹

DTI (targeting index) = the drug concentration in I tissue after administrating targeting preparation at T time/the drug concentration in I tissue after administrating non-targeting preparation at T time

DSI (selectivity index) = the drug concentration in target site at T time/the drug concentration in non-target site or blood at T time

DTE (targeting efficiency) = area under the concentration–time curve in target site/area under the concentration − time curve in non-target site or blood

RTE (relative targeting efficiency) = area under the concentration–time curve after administrating targeting preparation/area under the concentration–time curve after administrating non-targeting preparation

2.8 Statistical analysis

All of the experiments were repeated at least three times. Data are shown as the means standard deviation (SD). Student's t-test was used to identify significant differences. p values less than 0.05 were considered statistically significant.

3 Results and discussion

3.1 Synthesis of DSPE–PEG–cholic acid

Cholic acid was conjugated to DSPE–PEG through a reaction between the carboxy group and amino group is shown in Fig. 1. The conjugation reaction was monitored by HPLC. As shown in ESI Fig. S2†. The vivo fate of cholic acid after oral administration, the retention time for cholic acid was around 15 min and after 48 h reaction the peak of cholic acid almost disappeared, indicating that cholic acid had been successfully conjugated to the DSPE–PEG–NH₂. Mean molecular weight was determined by MALDI-TOF MS (ESI Fig. S3†), mean molecular weight of DSPE–PEG–NH₂ and cholic acid were at around 3000 and 408, respectively, the reaction product was mostly at around 3400, suggesting that the reaction product was mostly DSPE–PEG–cholic acid.

Fig. 1 Schematic illustrations of synthesis of DSPE–PEG–cholic acid.

3.2 Preparation and characterization of nanoliposomes

Silybin can be easily encapsulated into the hydrophobic core of nanoliposomes due to its hydrophobicity, the encapsulation efficiency (%, w/w) of silybin in both LPs–silybin and CA–LPs–silybin was over 95%. Both the functional nanoliposomes and unmodified nanoliposomes have very similar average diameters at about 100 nm with a polydispersity (PDI) less than 0.21 (Fig. 2A and ESI Table S1†), which may be an optimal size for gastrointestinal permeation and intracellular uptake. Moreover, the zeta potential analyses demonstrated that functional nanoliposomes exhibited lower negative surface charge than unmodified nanoliposomes, because the external PEG layer of functional nanoliposomes had no charge, it could shield negative charge of internal nanoliposomes to minimize the undesirable nonspecific protein adsorption. Transmission electron microscopy images showed the spherical shape of nanoliposomes (Fig. 2B and C).

Fig. 2 The characterization of modified nanoliposomes. (A) Particle size of CA–LPs–silybin by dynamic light scattering analysis (average size, D = 100 nm). Transmission electron microscopy images of LPs–silybin (B) and CA–LPs–silybin (C).

3.3 Receptor expression study

The expression of ASBT and NTCP in the Caco-2 and HepG2 cells is essential for the receptor-mediated endocytosis in our study. Therefore, it was examined and confirmed to be positive as shown in Fig. 3. Competitive inhibition experiments were conducted to verify our hypothesis that the increased cellular uptake was facilitated in the presence of ASBT and NTCP on cells. Significantly reducing expression of ASBT and NTCP was observed in the presence of cholic acid over time, indicating that ASBT and NTCP were saturated with cholic acid which may specifically interact with ASBT and NTCP.

Fig. 3 Cholic acid receptor expression in cells. (A) ASBT expression in Caco-2 cells (n = 3); (B) NTCP expression in HepG2 cells (n = 3). “**” indicates p < 0.01 versus the control. “^##” indicates p < 0.01 between LPs–silybin and CA–LPs–silybin. “Cholic acid 0.5 h” indicates cell monolayers were first incubated with cholic acid for 30 min. “Cholic acid 1 h” indicates cell monolayers were first incubated with cholic acid for 1 h.

3.4 Demonstration of the transport of nanoliposomes across the Caco-2 cell monolayer

3.4.1 Transport of nanoliposomes across the cell monolayer. As seen in Fig. 4, the transport of silybin rate: CA–LPs–silybin > LPs–silybin > silybin solution. The transport of silybin increased greatly after association it into the nanoliposomes and modified nanoliposomes, revealing the positive effect of nanoliposomes and cholic acid on hydrophobic molecules in terms of transport enhancement. To remove free drugs by the low speed centrifugation and ultrafiltration membrane filtration in basolateral chamber, measuring the content of drug in nanoliposome to ensure drug content loaded in the nanoliposomes. ESI Table S2† demonstrated the encapsulation potency of drugs in basolateral chamber were 89% and 96% for LPs–silybin and CA–LPs–silybin, respectively. The CA–LPs–silybin in basolateral chamber could keep integrity mostly.

Fig. 4 Transport of silybin across Caco-2 cell monolayer at 37 °C after incubation with silybin, LPs–silybin or CA–LPs–silybin for 2 h (n = 3). “**” indicates p < 0.01 versus the control; “^##” indicates p < 0.01 between LPs–silybin and CA–LPs–silybin.

3.4.2 The direct imaging of the nanoliposomes in apical and basolateral chamber by TEM and fluorescent images studies. The TEM image of apical and basolateral medium after incubation of transwell filter grown Caco-2 cell monolayer with nanoliposomes at 37 °C for 2 h were illustrated in Fig. 5A1, B1, A2 and B2, which indicated the functional nanoliposomes could traffick to the basolateral side after internalization. The size of particles in both apical and basolateral medium was almost the same. Fig. 5C revealed a portion of LPs–silybin's size increased, which demonstrated LPs–silybin aggregated after transport through Caco-2 cell monolayers, however, hydrophilic PEG in the surface of CA–LPs–silybin could prevent or delay the clustering of the nanoliposomes, so CA–LPs–silybin's size didn't increase in the basolateral medium. This method directly demonstrated the transport of the nanoliposomes across the cell monolayer, though it provides no information on drug loading in the nanoliposomes.

Fig. 5 TEM images of apical medium after incubation with (A1) LPs–silybin and (A2) CA–LPs–silybin for 2 h at 37 °C. TEM images of basolateral medium after incubation with (B1) LPs–silybin and (B2) CA–LPs–silybin for 2 h at 37 °C. Particle size distribution profiles of basolateral medium after incubation with (C1) serum free MEM, (C2) LPs–silybin, (C3) CA–LPs–silybin for 2 h at 37 °C, determined by dynamic light scattering.

3.5 Mechanistic studies on the transport of CA–LPs–silybin

3.5.1 Pathway studies with endocytosis inhibitors. The transcytosis process, involving membrane invagination, vesicle budding and movement of vesicles filled with cargo to the opposite side of the cell, begins by an uptake that depends on the type of endocytosis involved: macropinocytosis, clathrin mediated endocytosis, caveolae mediated endocytosis, and caveolae and clathrin independent endocytosis. The data in Fig. 6A indicated that the most effective inhibition in cell endocytosis of CA–LPs–silybin was from MβCD (p < 0.01), followed by nystatin (p < 0.01), chlorpromazine and genistein, and then NaN₃ and cholic acid (p < 0.01). It is reported that there are a lot of cholesterol-rich microdomains (lipid rafts) and flask shaped structures rich in proteins and lipids including cholesterol (caveolaes) in various cell membranes in different extents. The internalization of many extracellular macromolecules is reported to be mediated by lipid rafts or caveolaes via specific or non-specific interactions. MβCD and nystatin (cholesterol binding agents, known as inhibitors of caveolae/lipid raft-mediated endocytosis) could block the endocytosis process based on lipid raft/caveolae via the depletion of cholesterol of MβCD, reducing the cell uptake of CA–LPs–silybin significantly. Clathrin-mediated endocytosis is the classical endocyotis pathway of macromolecules and particles. Via the formulation of clathrin-coated pits, extracellular substances are loaded into cell membrane and internalized by cell along with those pits. Chlorpromazine could prevent clathrin mediated endocytosis by disrupting the assembly of the clathrin adaptor protein at the cell surface. Genistein could inhibit the activity protein complex amino acid kinase (PTK) and prevent r-phosphorylation on ATP to transfer to protein tyrosine residues. NaN₃ could inhibit ATP-dependent processes of cells. If ATP is depleted in cells, then active mechanisms are consequently inhibited. Therefore, the study here actually demonstrated the involvement of the cholesterol dependent, caveolae and clathrin mediated endocytosis of CA–LPs–silybin in Caco-2 cells. Cholic acid could reduce the transport of CA–LPs–silybin via saturating ASBT receptor, which demonstrated it had ASBT receptor mediated transport mechanism.

Fig. 6 Transport of CA–LPs–silybin across Caco-2 cell monolayer. (A) Papp of CA–LPs–silybin transport in the presence of respective inhibitors (n = 3); (B) Papp of CA–LPs–silybin transport with or without EGTA treatment (control) (n = 3); (C) effect of temperature on CA–LPs–silybin' transport rate. Values indicate means ± SD (n = 3). “**” indicates p < 0.01 versus control. “**” indicates p < 0.01 versus control. “☆☆” indicates p < 0.01 between 22 °C and 4 °C.

3.5.2 Effect of EGTA on the transport. The pore diameter of intercellular space of gastrointestinal epithelial cells was less than 10 Å, its absorption area accounted for only 0.1% of the surface of intestinal mucosa, LPs couldn't pass through the epithelial cells by paracellular channel largely. In order to investigate the possible paracellular pathway of CA–LPs–silybin, its transport across Caco-2 cell monolayers treated and not treated with 3 mM EGTA solution were compared. It is well known that Ca²⁺ ions have an important role to maintain the paracellular permeability of epithelial cell monolayers by modulating the tight junctions. EGTA pretreatment upsets cell polarity by chelating calcium and thus disrupts existing tight junctions. In this test, opening the tight junctions, induced by EGTA, had no obvious increase in Papp of silybin, as illustrated in Fig. 6B. In other words, this barrier of the cell monolayer indicates that the transport of nanoliposomes was mainly through the transcellular pathway.

3.5.3 Effect of other factors on the transport. Fig. 6C showed the effects of temperature on the transport rates. As we know, all ATP-dependent processes of cells will be inhibited at 4 °C. If ATP is depleted in cells, then active mechanisms are consequently inhibited. As seen in Fig. 6C, lowering the temperature from 37 °C to 22 °C or 4 °C significantly reduced the values of Papp of CA–LPs–silybin, indicating that a great extent of transport is temperature dependent or energy dependent.

3.6 Endocytosis pathways of CA–LPs–silybin

3.6.1 In vitro cellular uptake studies. The cellular uptake of LPs–silybin and CA–LPs–silybin was first evaluated in hepatocellular carcinoma cell HepG2. Based on HPLC and BCA analysis (Fig. 7A), the cellular silybin level was significantly higher from the CA–LPs–silybin group relative to free silybin and LPs–silybin groups, demonstrating that cholic acid modification significantly increased the endocytosis of nanoliposomes. It was suggested that the cholic acid could markedly improve the recognition and uptake of nanoliposomes when it is bonded on their surface. Fig. 7B shows fluorescence microscopy photographs of HepG-2 cells exposed to pure DIO, LPs-Dio or CA–LPs–Dio. The fluorescence intensity of DIO for DSPE–PEG–cholic acid nanoliposomes was much higher than that of unmodified nanoliposomes and pure DIO, which confirmed that CA–LPs were taken up more efficiently by HepG2 cells, resulting in higher efficacy drug molecules delivery.

Fig. 7 Silybin uptake by HepG2 cell at 37 °C (A). (A) Uptake from different formulations of silybin (n = 3); (B) Laser scanning confocal microscopy images of the HepG2 cells incubated with DIO, LPs-Dio and CA- LPs-Dio; (C) effect of inhibitors on the uptake of CA–LPs–silybin (n = 3); (D) effect of temperature on CA–LPs–silybin' uptake rate (n = 3). “**” indicates p < 0.01 versus control. “^##” indicates p < 0.01 between LPs–silybin and CA–LPs–silybin. “☆☆” indicates p < 0.01 between 22 °C and 4 °C.

3.6.2 Endocytosis pathway detection by uptake inhibitors. During the uptake of CA–LPs–silybin with HepG2 cells, various endocytosis inhibitors were utilized to identify the endocytosis pathways. As shown in Fig. 7C, the inhibition effect of MβCD on cellular uptake of CA–LPs–silybin was most obvious as compared to other inhibitors used in this study, followed by genistein (p < 0.01), chlorpromazine and NaN₃, and then nystatin (p < 0.01). The inhibition mechanism of MβCD, chlorpromazine, genistein, NaN₃ and nystatin were the same as“3.5.1”. Cholic acid could reduce uptake via saturating NTCP receptor, which demonstrated that the CA–LPs–silybin could be endocytosed via NTCP mediated endocytose by HepG2 cells, involving multiple pathways (both lipid raft and clathrin mechanisms), which was an energy-dependent process.

3.6.3 Effect of other factors on the cellular uptake. As we know, all ATP-dependent processes of cells will be inhibited at 4 °C. If ATP is depleted in cells, then active mechanisms are consequently inhibited. As seen in Fig. 7D, lowering the temperature from 37 °C to 22 °C or 4 °C significantly reduced the uptake of CA–LPs–silybin, indicating that a great extent of uptake is temperature dependent or energy dependent.

3.7 In vivo targeting efficiency

In vivo hepatic distribution studies was carried out to evaluate whether the entrapment of silybin into the functional nanoliposomes could increase the bioavailability and liver targeting specificity of the silybin. Oral administration route was chosen to carry out these experiments as it is the best route in terms of patient compliance and silybin is currently administered in patients as tablet or capsule dosage forms. In the field of oral drug delivery, drug encapsulation into polymeric carriers such as nanoliposomes could overcome several bioavailability-reducing drawbacks usually associated with oral administration. In particular, encapsulation could protect sensitive drugs from degradation in stomach and gut lumen, enhance absorption in the intestine by increasing water solubility, overcome drug resistance mechanisms (MDR) by altering the absorption pathway from transcellular to paracellular or transcytosis routes. Moreover, the capability of some polymers to increase the drug adsorption through the gastro-intestinal mucosa either by increase the membrane permeability to the drug and/or to the carrier has been also reported.

In our experiments, three formulations were used for the treatment: the silybin solution and silybin encapsulated in LPs (LPs–silybin) and CA–LPs (CA–LPs–silybin). These formulations were orally administered by gastric gavage, after dispersion in the vehicle (isotonic saline solution) at the single dose of 100 mg kg⁻¹ of the body weight. After 0.25, 0.5, 1, 1.5, 2 and 4 h post administration, the concentration of silybin in liver and blood was determined by HPLC analysis as described in the experimental section.

In the blood, the mean silybin concentration after oral administration of a single dose of silybin, LPs–silybin or CA–LPs–silybin was illustrated in Fig. 8A. The concentration–time data of the three preparations were best fitted to a two-compartment model, and the relevant pharmacokinetic parameters such as C_max, T_max, AUC0-240 and mean residence time (MRT) were given in ESI Table S3.† The results showed that there was no significant difference between the pharmacokinetic parameters of the mean silybin concentration in the blood after oral administration of a single dose as free drug (silybin) or as non-targeted nanoliposomes (LPs–silybin). However, significantly higher concentration was observed with the targeted nanoliposomes (CA–LPs–silybin) as compared with the first two formulations (p < 0.05 and p < 0.01, respectively).

Fig. 8 Mean silybin concentration in blood following oral administration of different formulations, mean F S.E. (n = 6) (A). Mean silybin concentration in liver following oral administration of different formulations, mean F S.E. (n = 6) (B).

In the liver, the silybin concentration at 4 h post administration (Fig. 8B) was significantly higher with the targeted nanoliposomes (CA–LPs–silybin) as compared with non-targeted nanoliposomes (LPs–silybin, p < 0.01) or free drug (silybin, p < 0.01). The pharmacokinetic data of liver were given in ESI Table S4.† The oral hepatic targeted parameters of different formulations were shown in Table 1, which demonstrated that the drug in targeted nanoliposomes reached the liver to a significantly larger extent than the drug administered as free drug or loaded into non-targeted nanoliposomes. The significantly increased liver accumulation associated with targeted nanoliposomes could allow a reduction of administered dose. All the above results suggest that the use of CA–LPs–silybin increased the liver targeting of silybin via NTCP mediated internalization.

Table 1 Oral hepatic targeted parameters of different formulations of silybin (n = 6)^a

Groups	DTI (30 min)	DSI (30 min)	DTE	RTE
a Comparative oral hepatic targeted parameters of silybin solution, LPs–silybin and CA–LPs–silybin 4 h post administration. (Mean ± S.E., n = 6, **p < 0.01 between silybin solution and other formulations, ^##p < 0.01 between LPs–silybin and CA–LPs–silybin).
Free drug		2959.98	2727.64
LPs–silybin	^##3.51	**^##10 520.24	**^##5380.43	^##2.16
CA–LPs–silybin	^##10.99	**^##24 032.63	**^##8972.15	^##4.35

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4 Conclusions

This study demonstrated that the cholic acid receptor-targeted functional nanoliposomes increased the intestine transport and liver intracellular uptake, via enhancing their transcytosis in polarized Caco-2 cells and uptake in HepG2 cells. In vitro transport and uptake experiments showed a higher uptake and transport of CA–LPs–silybin over LPs–silybin via the specific binding of cholic acid on these nanoliposomes to the receptors on the cell membrane. Endocytosis pathway experiments indicated that CA–LPs–silybin entered the cells through both lipid raft and clathrin mechanisms related to the expression of ASBT on Caco-2 cells and NTCP on Caco-2 cells. It is therefore concluded that the cholic acid modified nanoliposomes could specifically interact with GI endothelial cells and liver cells and result in increased cellular uptake, transcytosis and hepatic distribution (ESI Fig. S4†). Hepatic distribution data demonstrated that the amount of silybin delivered by drug loaded functional nanoliposomes systems to the liver in significantly greater extent than by both free silybin solution and silybin-loaded non-targeted nanoliposomes. Therefore, the presence of cholic acid on DSPE–PEG–cholic acid-nanoliposomes confers them the capability of enhanced accumulation of silybin into the liver relative to non-cholic acid systems, demonstrating the potential contribution of cholic acid receptor to the oral hepatic targeted process.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 81274094) and the Graduate Student Innovation Fund of Peking Union Medical College (No. 10023-1007-1017).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra28018d

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