Evaluation of the Combined Effect of Recombinant High-Density

Evaluation of the Combined Effect of Recombinant High-Density Lipoprotein. 1. Carrier and the Encapsulated Lovastatin in RAW264.7 Macrophage Cells Bas...
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Article Cite This: Mol. Pharmaceutics 2018, 15, 1017−1027

Evaluation of the Combined Effect of Recombinant High-Density Lipoprotein Carrier and the Encapsulated Lovastatin in RAW264.7 Macrophage Cells Based on the Median-Effect Principle Cuiping Jiang, Yi Zhao, Yun Yang, Jianhua He, Wenli Zhang,* and Jianping Liu* Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210008, PR China S Supporting Information *

ABSTRACT: Recombinant high-density lipoprotein (rHDL) displays a similar anti-atherosclerotic effect with native HDL and could also be served as a carrier of cardiovascular drug for atherosclerotic plaque targeting. In our previous studies, rHDL has shown a more potent anti-atherosclerotic efficacy as compared to the other conventional nanoparticles with a payload of lovastatin (LS). Therefore, we hypothesized that a synergistic anti-atherosclerotic effect of the rHDL carrier and the encapsulated LS might exist. In this study, the dose−effect relationships and the combined effect of the rHDL and LS were quantitatively evaluated in RAW 264.7 macrophage cells using the median-effect analysis, in which the rHDL carrier was regarded as a drug combined. Median-effect analysis suggested that rHDL and LS exerted a desirable synergistic inhibition on the oxLDL internalization at a ratio of 6:1 (Dm,LS:Dm,rHDL) in RAW 264.7 macrophage cells. About 50% of the reduction on the intracellular lipid contents was found when RAW264.7 cells were treated with LS-loaded rHDLs at their respective median-effect dose (Dm) concentrations and a synergistic effect on the mediating cholesterol efflux was also observed, which verified the accuracy of the results obtained from the median-effect analysis. The mechanism underlying the synergistic effect of the rHDL carrier and the drug might be attributed to their potent inhibitory effects on SR-A expression. In conclusion, the median-effect analysis was proven to be a feasible method to quantitatively evaluate the synergistic effect of the biofunctional carrier and the drug encapsulated. KEYWORDS: recombinant high-density lipoprotein, median-effect principle, synergistic effect, drug combination, atherosclerotic targeting and foam cells.4,11 Therefore, rHDL may serve as a biofunctional drug delivery candidate of cardiovascular drugs. Lovastatin (LS), a member of the 3-hydroxy-3-methylglutaryl−coenzyme A (HMG-CoA) reductase inhibitors, is widely used as an anti-atherosclerotic drug.12,13 Besides its blood lipid lowering effect, LS plays an important role in attenuating vascular plaque inflammation,14 which is ascribed to an interplay of pleiotropic effects on endothelial dysfunction, oxidative stress, and thrombosis.15 In our previous studies, the rHDL carrier has been utilized with a payload of LS for the improved anti-atherosclerotic therapy.16,17 The results demonstrated that LS-loaded rHDLs (LS-rHDLs) had higher plaque targeting efficiency and more potent anti-atherogenic efficacy in an atherosclerotic rabbit model compared with that of the other LS-loaded nano drug delivery systems.17 Therefore, a synergistic anti-atherosclerotic effect of LS and rHDL carrier

1. INTRODUCTION High-density lipoprotein (HDL) is an important plasma lipoprotein in the lipid transport system,1 whose level is considered to be inversely correlated with the incidence of atherosclerosis (AS).2 The antiatherogenic activities of HDL are mainly attributed to reverse cholesterol transport (RCT),3 during which HDL is capable of removing excessive cholesterol from plaque macrophages and foam cells to the liver for biliary excretion through the interaction of apoA-I with a scavenger receptor BI (SR-BI), ATP-binding cassette transporter A1 (ABCA-1), and G1 (ABCG-1) receptors.4 Along with RCT, HDL possesses a multitude of other cardiovascular protective effects, including the antioxidative effect, the endothelial protective function, as well as the antithrombotic property.5 Several lines of evidence have shown that recombinant HDL (rHDL) possesses an atheroprotective effect similar to the native HDL.6,7 Recently, rHDL has gained increasing attention as a drug vehicle due to favorable attributes, including lipid space for hydrophobic drugs, long circulation time in blood, and the capacity to evade the reticuloendothelial system, etc..8−10 Besides, rHDL could target the atherosclerotic plaque via the overexpressed SR-BI receptor in plaque macrophages © 2018 American Chemical Society

Received: Revised: Accepted: Published: 1017

October 23, 2017 January 23, 2018 January 30, 2018 January 30, 2018 DOI: 10.1021/acs.molpharmaceut.7b00923 Mol. Pharmaceutics 2018, 15, 1017−1027

Article

Molecular Pharmaceutics

method and the subsequent incubation of the liposome with an apoA-I mediated by a sodium cholate for the rHDL formation as previously depicted with minor modification.17,27 The specific methods were described as follows. Liposome was prepared by a thin film dispersion method. Specifically, 0.2 g of soy lecithin and 0.02 g of cholesterol were dissolved in 15 mL of chloroform/methanol (1:1, v/v) and then dried in an egg-plant flask under vacuum at 40 °C for 1 h. The same volume (15 mL) of Tris-HCl buffer (pH 8.0) was added to hydrate the film, and then the suspension was sonicated thoroughly for 10 min in an ice bath at 300 W with an Ultra-Homogenize (JY92II; Ningbo, China). The resultant suspension was filtered through a 0.22 μm filter to remove large particles. The liposome obtained above was further incubated with apoA-I induced by sodium cholate to form the rHDL carrier. To achieve the desirable apoA-I coupling efficiency for rHDL, 2 mg/mL of apoA-I was confirmed in the preparation of rHDL by a fluorescence quenching experiment in our preliminary study. Briefly, 5 mL of liposome was incubated with 10 mg of apoA-I and 20 mg of sodium cholate under the rotating speed of 300 rpm at 4 °C for 8 h. After incubation, the rHDL solution was dialyzed against Tris-HCl buffer (pH 8.0) overnight to remove the excess sodium cholate. 2.2.2. Preparation of LS-rHDLs. LS-rHDLs were formulated with varied LS/rHDL ratios upon their respective median-effect doses (Dm) as mentioned in the Evaluation of LS-rHDLs Based on the Median-effect Principle Section. Different ratios of LS/ rHDL upon their respective Dm values ranging from the low level to the high level were chosen in the preparation of LSrHDLs in our preliminary study. To ensure the high drug entrapment efficiency and the favorable stability, LS-rHDLs were formulated with the ratios of LS/rHDL at 3:1, 6:1, and 9:1 (Dm,LS:Dm,rHDL). Since the Dm values of the LS and rHDL carrier were quite low, the amounts of LS and the feeding materials of rHDLs were amplified at the same extent in the preparation process of LS-rHDLs. Similarly, the methods of LS-rHDLs were identical to that of the rHDL carrier except for adding different amounts of LS in the formulations of liposomes. Specifically, 5, 10, and 15 mg of LS were added in the formulation of liposome to prepare different LS-liposomes, respectively. LS-rHDLs were then obtained through incubation of LS-liposomes with apoA-I mediated by sodium cholate. 2.3. Physicochemical Characterization of the LSrHDLs. 2.3.1. Mean Size, Zeta Potential, Drug Entrapment Efficiency (EE), and Drug Loading (DL). The mean sizes and zeta potential of the LS-liposome and LS-rHDL were determined by a dynamic light scattering (DLS) analyzer (Zetasizer 3000 HAS, Malvern, UK). All samples were diluted appropriately with aqueous phase prior to the measurements. EE and DL of preparations were measured after the elimination of nonentrapped drug by the mini-column centrifugation method as previously described.8,27 Then, the amounts of LS entrapped in LS-liposome and LS-rHDL were quantified by HPLC (Agilent Technologies, Palo Alto, CA, USA) equipped with an ultraviolet detector conducted at 238 nm and a shim-pack VP-ODS column (150 × 4.6 mm,5 μm) at the temperature of 30 °C. The mobile phase was made up of methanol and deionized water (80:20, v/v), and the flow rate was kept at 1.0 mL/min. EE and DL were calculated according to the following equations.

may exist. As such, investigating their dose−effect relationships would be beneficial for the evaluation of their combined effect. The median-effect principle, based on the mass-action law, is a general theory of dose−effect relationship.18 The combination index (CI) derived from the median-effect equation is an effective scientific index to depict the combined effect of two or multiple compounds, where CI > 1, = 1, and < 1 represent antagonism, additive effect, and synergism, respectively.18,19 It is recommended that the lower CI value indicates the stronger synergistic effect. The median-effect analysis has been widely applied for the evaluation of two drug combinations.20,21 For example, Chih-Chuan Chang et al. manifested that a combination of tetra-O-methyl nordihydroguaiaretic acid and paclitaxel would produce a synergistic anticancer effect in nude mice based on the median-effect principle.22 Kathryn M. Camacho and co-workers utilized the median-effect analysis to explore the synergistic ratios of doxorubicin and 5-fluorouracil for the low dose chemotherapy in the PEGylated liposome system.23 However, to the best of our knowledge, the combined effect of the biofunctional carrier and the drug encapsulated so far has been rarely reported. In this study, the median-effect analysis was employed to investigate the combined effect of rHDL and the encapsulated LS. In view of the atheroprotective effect of the rHDL carrier,24 it was regarded as a drug combined. The inhibitory effects of LS and rHDL on DiI-oxLDL internalization in RAW 264.7 cells were measured. Their dose−effect relationships were quantitatively established and the combined effect was evaluated by the CI value based on the median-effect principle. In order to verify the accuracy of the results obtained from the median-effect analysis, intracellular lipid contents and cholesterol efflux capability were investigated. Besides, the mechanism underlying the synergistic effect of LS and rHDL was studied by Western blotting. Furthermore, in vitro physicochemical properties of LS-rHDLs were characterized in terms of the mean size, zeta potential, morphology, drug entrapment efficiency, and drug loading efficiency. Moreover, the cellular uptake and targeting mechanism of LS-rHDL were investigated.

2. MATERIALS AND METHODS 2.1. Materials and Reagents. Phospholipids (Lipoid S100) were obtained from Lipoid GmbH (Germany). Cholesterol, MTT, and oil red O were purchased from Sigma-Aldrich Chemie GmbH Co. Ltd. Glycerol trioleate was purchased from Tokyo Kasei Kogyo Co. Ltd. (Japan). Cholesterol oleate was a product of Alfa Aesar/Johnson Matthey Co. Ltd. (UK). OxLDL and DiI-oxLDL were supplied by Yiyuan Biotechnology (Guangzhou, China). Radio immunoprecipitation assay (RIPA lysis buffer), phenylmethanesulfonyl fluoride (PMSF), and BCA protein assay kit were products of Beyotime Biotechnology (Shanghai, China). The apoA-I (97% purity) was isolated from the industrial waste of albumin as described previously with minor modification.25 Sephadex G50 was purchased from Pharmacia (Sweden). All reagents were of analytical or chromatographic grade. Distilled and deionized water was used in all experiments. Lovastatin (LS), the inactive lactone form, was kindly donated by Jiangsu Yangzi River Pharmacy Company (Taizhou, Jiangsu, China). It was converted into the active form in accordance with the method Liang et al. described.26 2.2. Preparation of rHDL Carrier and LS-rHDLs. 2.2.1. Preparation of rHDL Carrier. The preparation procedure consisted of constructing the liposome by a thin film dispersion 1018

DOI: 10.1021/acs.molpharmaceut.7b00923 Mol. Pharmaceutics 2018, 15, 1017−1027

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Molecular Pharmaceutics EE(%) =

W × 100% W0

(1)

DL(%) =

W × 100% Wt

(2)

within the safe concentration ranges tested by cytotoxicity assays. 2.6.1. DiI-oxLDL Uptake by Macrophages. RAW264.7 cells were plated at the concentration of 2 × 105 cells per well in 12well plates and maintained in DMEM with 10% (v/v) of FBS and 1% (v/v) of penicillin:streptomycin. Cells from each group were seeded in triplicate. After 12 h of incubation, the culture medium was replaced with LS solution (0, 0.004, 0.02, 0.04, 0.2, 0.4, 2.0, 4.0 μg/mL), rHDL carrier (0, 100, 200, 400, 600, 700 μg/mL), or different LS-rHDLs containing DiI-oxLDL (80 μg/ mL), respectively. After exposure for 24 h, the cells were rinsed with PBS (pH 7.4) to remove the nonphagocytized DiI-oxLDL. Then, the cells were harvested by centrifugation at 5000 rpm for 10 min and washed thrice with PBS (pH 7.4). The MFI of DiI-oxLDL in each group was determined by flow cytometry. 2.6.2. Evaluation of Drug Combinations. In this study, the median-effect analysis of Chou-Talalay18 was employed to evaluate the combined effect of LS and rHDL carrier. Specifically, the dose−effect relationships of LS and rHDL were established to acquire their respective Dm values by the median-effect principle. Then, LS-rHDLs were formulated with the varied ratios of LS/rHDL upon their respective Dm values. The combined effect was determined by the CI values. The combined parameters and graphs were analyzed by the CompuSyn software program (version 3.0, ComboSyn Inc., Paramus, NJ). The calculations of this program are listed step by step as follows.18,30,31 Step 1: Dose−effect relationships of LS and rHDL were analyzed by the following median-effect equation described by Chou.18 LS and rHDL were regarded as drug 1 and drug 2, respectively.

where W means the amount of LS entrapped in LS-liposome or LS-rHDL, W0 means the total amount of LS added in the formulation of LS-liposome or LS-rHDL, and Wt means the total amount of the feeding materials. 2.3.2. Transmission Electron Microscopy (TEM). Morphological examinations of LS-liposome and LS-rHDL were carried out by TEM (H-7650, Hitachi High-Technologies Corporation, Japan). In brief, a drop of each preparation stained with 2% (w/ v) of phosphotungstic acid solution was placed onto a copper grid coated with a carbon film and then air-dried under ambient temperature. 2.4. Cellular Uptake and Targeting Mechanism Study of rHDL. To identify the targeting effect of rHDL in mouse macrophage cell line RAW264.7 cells, fluorescent coumarin-6 (C6) was incorporated into rHDL with the same method as LS-rHDL, except for the replacement of LS by C6.28 RAW264.7 cells were plated at the concentration of 1 × 105 cells per well in 12-well plates and maintained in DMEM with 10% (v/v) of FBS and 1% (v/v) of penicillin:streptomycin until the 80% confluence. Then the culture medium was replaced with C6-loaded liposome (C6-liposome) or C6-loaded rHDL (C6-rHDL), respectively. After exposure for 24 h, the cells were rinsed with PBS (pH 7.4) thrice and harvested by centrifugation at 5000 rpm for 10 min. The mean fluorescence intensity (MFI) of C6 in each group was determined by flow cytometry. In order to study the targeting mechanism of rHDL, Raw 264.7 cells were preincubated with a selective inhibitor of SR-BI (block lipid transport 1, BLT-1) or the excess apoA-I at the concentrations of 100 μM or 50 mg/mL,29 respectively. After treatment for 2 h, cells were washed thrice with PBS (pH 7.4) and then incubated with C6-liposome and C6-rHDL for 24 h. Then, cells were washed, collected, and resuspended with PBS. The MFI of C6 in each group was determined by flow cytometry. 2.5. Cytotoxicity Assays. The cytotoxicities of LS solution, rHDL carrier, and LS-rHDLs were examined by MTT assay. RAW264.7 cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) of fetal bovine serum (FBS) and 1% (v/v) of penicillin:streptomycin were seeded in 96-well plates (Costar, Corning, NY) at a density of 1 × 104 cells per well in a humidified atmosphere containing 5% CO2. After overnight culturing, cells were treated with LS (1−20 μM), rHDL (100−1800 μg/mL), and different LS-rHDLs (300 μg/mL) for 24 h, respectively. After washing thrice with PBS (pH 7.4), 200 μL of MTT (0.5 mg/mL) was added to each well, and the cells were further cultured for 4 h. Then, 150 μL of dimethyl sulfide (DMSO) was added to each well, and the OD value was measured at 570 nm with a microplate reader (Biotek ELx800). 2.6. Evaluation of LS-rHDLs Based on the MedianEffect Principle. To evaluate the combined effect of LS and rHDL carrier, their dose−effect relationships were established first. The inhibitory effect on uptake of fluorescently labeled DiI-oxLDL in RAW264.7 cells was chosen as a drug effect index. The dose ranges and dose densities of LS, rHDL carrier, and LS-rHDLs in this part of the experiments were selected

fa /fu = (D/Dm)m

(3)

where D is dose, Dm is the dose required to produce the median-effect (e.g., 50% of inhibition on DiI-oxLDL uptake, corresponding to ED50), and m is the Hill-type coefficient signifying the shape of the dose−effect curve. Where fa and f u are the fractions of cells affected and unaffected, respectively. On the basis upon the logarithmic form of the median-effect equation log(fa /fu ) = m log D − m log Dm

(4)

The above linear dose−effect curves could be expressed as a classic straight line equation y = mx + b

(5)

where x = log D, y = log ( fa/f u), b = −mlogDm, and m is slope. The Dm value was calculated from the antilog of the −y intercept/ m. In our studies, fa was explained by the following equation. fa = 1 − MFI/MFIcontrol

(6)

where MFI and MFIcontrol are the mean fluorescence intensities of the drug-treated working groups and the untreated control group, respectively. Step 2: Then, LS-rHDLs formulated with the varied ratios (i.e., 3:1, 6:1, and 9:1 (Dm,LS:Dm,rHDL)) were evaluated by the median-effect analysis. The parameters m and Dm of LS-rHDLs were obtained from the median-effect plot as described in step 1. These parameters composed the following equation: 1019

DOI: 10.1021/acs.molpharmaceut.7b00923 Mol. Pharmaceutics 2018, 15, 1017−1027

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Molecular Pharmaceutics Table 1. Particle Size, Zeta Potential, EE, and DLa preparations

ratios (Dm,LS:Dm,rHDL)

LS-liposome

3:1 6:1 9:1 3:1 6:1 9:1

LS-rHDL

a

size (nm) 112.5 114.8 115.3 102.2 103.1 104.9

± ± ± ± ± ±

0.5 0.9 1.9 1.3b 0.7b 2.8b

zeta potential (mV) −20.6 −24.3 −21.1 −31.4 −35.7 −34.2

± ± ± ± ± ±

2.2 3.4 1.6 1.51b 2.44b 1.82b

EE (%)

DL (%)

94.26 95.77 93.52 94.39 93.74 91.80

2.01 3.99 5.73 1.78 3.47 5.01

Mean value ± SD, n = 3. bSignificant differences between LS-liposome and LS-rHDL at the same Dm,LS:Dm,rHDL ratios (p < 0.05).

Dx = Dm × [fa /(1 − fa )]1/ m

2.7.3. Cholesterol Efflux Assay. RAW 264.7 cells seeded in 6-well plates were treated with oxLDL (80 μg/mL) for 24 h and then incubated with NBD-cholesterol (5 μM) for 24 h. After being washed thrice with PBS, the cells were incubated with LS, rHDL, LS-liposome, and 6:1 (Dm,LS:Dm,rHDL) LSrHDL for an additional 24 h.32 Then the cell medium was collected and centrifuged at 5000 rpm for 5 min at 4 °C. The cells were lysed in RIPA buffer and the cell lysates were centrifuged at 10000 rpm for 10 min at 4 °C. The fluorescent intensities of NBD-cholesterol in the medium (Fm) and the cell lysates (Fc) were measured with a microplate reader. The percentage of cholesterol efflux was calculated according to the formula: Fm/(Fm + Fc) × 100%. 2.8. Synergistic Mechanism of LS and rHDLs by Western Blotting. RAW 264.7 cells plated in 6-well plates stimulated with oxLDL (80 μg/mL) were treated with 6:1 (Dm,LS:Dm,rHDL) LS-rHDL for 24 h. After being washed thrice with PBS, the cells were lysed in RIPA buffer with 1% of freshly added PMSF for 2 h on ice. Protein concentrations were measured using the pierce BCA protein assay kit. Then, 40 μg of denatured proteins were subjected to 10% (w/v) SDSpolyacrylamide gel. Following the electrophoresis, proteins in SDS-polyacrylamide gel were transferred to PVDF membranes. Then, the PVDF membranes were blocked for 2 h followed by probing with antibodies (Abcom) for SR-A, CD36 (diluted 1:5000 in TBST), and rabbit anti-actin (diluted 1:1000 in TBST) overnight at 4 °C. Then, PVDF membranes were incubated with secondary antibodies for 2 h at room temperature. The signals were detected with the ECL system and analyzed by Quality one software v4.62. 2.9. Statistical Analysis. Data were expressed as mean ± SD from at least three independent experiments. Significant differences were determined using two-tailed paired Student’s t test or one-way analysis of variance (ANOVA), where differences were considered significant (*p < 0.05 and **p < 0.01).

(7)

where Dx is the dose (drug 1, drug 2, or drug 1 + drug 2) required to achieve x% effect. Step 3: In order to evaluate the combined effect of LS and rHDL, the combination index (CI) derived from the medianeffect equation was considered as an effective scientific index. The CI equation for the two drugs combination was described as follows. CI =

(D)1 (D)2 γ(D)1(D)2 + + (Dx )1 (Dx )2 (Dx )1(Dx )2

(8)

where (D) 1 or 2 represents the dose of drug1 or 2 required to produce the x% effect when the two drugs are used in combination, (Dx)1 or 2 is the dose of drug1 or 2 required to produce x% effect when the two drugs are used separately. Where CI > 1, = 1, and < 1 indicated antagonism, additive effect, and synergism, respectively. If the effect of two drugs was mutually nonexclusive, then γ = 1. If the effect of two drugs was exclusive, then γ = 0. The dose reduction index (DRI) was applied to evaluate the folds of dose reduction for each drug when the two drugs were used in combination. DRI can be defined as follows. DRI =

(Dx ) (D)

(9)

2.7. Validation of the Results of Median-Effect Analysis. 2.7.1. Determination of Intracellular Cholesterol Esters Content (CE). Raw 264.7 cells plated in 6-well plates stimulated with oxLDL (80 μg/mL) were treated with different LS-rHDLs at a concentration of their respective Dm values for 24 h. After being washed thrice with PBS (pH 7.4), the protein of the cells was isolated by lysis buffer followed by the measurement of the protein concentration using the pierce BCA protein assay kit. A cholesterol quantification kit was then utilized according to the manufacturer’s protocol to quantify the total cholesterol (TC) and free cholesterol (FC). CE was expressed as the difference between TC and FC. 2.7.2. Intracellular Lipid Droplets Stained by Oil Red O. Raw 264.7 cells plated in 24-well plates stimulated with oxLDL (80 μg/mL) were treated with different LS-rHDLs at a concentration of their respective Dm values for 24 h. Then, cells were washed thrice with PBS and fixed in paraformaldehyde (4%) for 30 min. After that, cells were rinsed with PBS thrice and stained with oil red O working solution for 30 min in darkness and then were washed with 60% of isopropanol. After being rinsed with PBS for 3 min, cells were dispersed in PBS and then visualized by the microscope. Intracellular lipid deposition was indicated by the positive staining area (%), which was analyzed by the Image-Pro Plus 6 software.

3. RESULTS 3.1. Physicochemical Characterization of LS-rHDLs. 3.1.1. Mean Size, Zeta Potential, EE, and DL. Table 1 shows that after incubation with apoA-I, the mean diameters of LSrHDLs were decreased (p < 0.05) compared with that of LSliposomes. Besides, the negative surface charge of liposomes was significantly (p < 0.05) increased after incubation with apoA-I. Moreover, the EE of LS-liposomes and LS-rHDLs was higher than 90% with desirable DL. 3.1.2. Visualization by Transmission Electron Microscope (TEM). Since LS-rHDLs formulated with the different ratios of LS/rHDL upon the D m values possessed the similar physicochemical properties, LS-rHDL formulated with 6:1 (Dm,LS:Dm,rHDL) and the corresponding LS-liposome were visualized by TEM. As depicted in Figure 1, LS-liposome 1020

DOI: 10.1021/acs.molpharmaceut.7b00923 Mol. Pharmaceutics 2018, 15, 1017−1027

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Molecular Pharmaceutics

Conversely, the uptake of liposomes showed no significant difference neither with BLT-1 nor apoA-I treatment (p > 0.05). 3.3. Cytotoxicity Assays. As shown in Figure 3, the cytotoxicity result of LS illustrated that the cells viability rates

Figure 1. TEM images of LS-liposome (A) and 6:1 (Dm,LS:Dm,rHDL) LS-rHDL (B).

(Figure 1A) was in spherical shape. However, the liposome was transformed from vesicle into discoidal stack after incubation with apoA-I (Figure 1B). These results were consistent with our previous studies.33 3.2. Cellular Uptake and Targeting Mechanism Study of rHDL. As exhibited in Figure 2, the cellular uptake of C6rHDL was about 1.8-fold higher than that of the C6-liposome (p < 0.01). In the study of the targeting mechanism of rHDL, the uptake of rHDL was decreased by 55.3 or 43.9% after blockage of SR-BI by BLT-1 or the excess apoA-I, respectively.

Figure 3. In vitro cytotoxicities of rHDL carrier (up) and LS (down) in RAW 264.7 cells (mean value ± SD, n = 6).

Figure 2. Cellular uptake and targeting mechanism in RAW264.7 cells investigated by flow cytometry analysis. **, p < 0.01 (mean value ± SD, n = 3). 1021

DOI: 10.1021/acs.molpharmaceut.7b00923 Mol. Pharmaceutics 2018, 15, 1017−1027

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Molecular Pharmaceutics

different LS-rHDLs were much lower than that of the rHDL carrier, inferring LS possessed the potent drug efficacy. Moreover, the r values of different preparations were all close to 1. Figure 5 shows the combined effect plots of LS-rHDLs. The results demonstrated that the CI values increased with the increment of effect levels (fa values), suggesting the synergic effect occurred at low fa levels and the antagonistic effect occurred at high fa levels. Therefore, LS-rHDLs formulated with the varied ratios of LS/rHDL at 3:1, 6:1, and 9:1 (Dm,LS: Dm,rHDL) were evaluated at the low ranges of effect levels with three representative points (i.e., ED40, ED45, ED50). On account of the lowest CI value, the best synergistic effect was observed when LS-rHDL was formulated with LS/rHDL at a 6:1 ratio upon the Dm values (Figure 5B). This preparation possessed the widest synergistic range (0 < fa < 0.6) and the highest DRI values (Table S1). Its Dm value was 30.82 μg/mL, and the CI values at ED40 ( fa = 0.4), ED45 (fa = 0.45), and ED50 ( fa = 0.5) were 0.61, 0.67, and 0.76, respectively (Table 2). However, LSrHDL formulated with LS/rHDL at a 3:1 ratio upon Dm values exhibited a synergistic effect when fa values were lower than 0.4 (Figure 5A). A synergistic effect was found in LS-rHDL when it was formulated with LS/rHDL at a ratio of 9:1 (Dm,LS:Dm,rHDL) at low fa levels ranging from 0 to 0.55 (Figure 5C). 3.5. Validation of the Results of Median-Effect Analysis. In order to verify whether the results obtained from the median-effect analysis were accurate, lipid contents of RAW 264.7 cells treated with different LS-rHDLs at their respective Dm concentrations were measured by a cholesterol quantification assay and oil red O staining. Besides, cholesterol efflux assay was performed to further validate the synergistic effect of LS-rHDL. 3.5.1. Determination of Intracellular Cholesterol Esters (CE) Content. Figure 6 depicts the intracellular CE contents of foam cells when the cells were treated with different LS-rHDLs at their respective Dm concentrations. The results demonstrated that LS-rHDLs-treated groups were all reduced by about 50% of intracellular CE contents as compared to that of the positive control group (group E). There was no significant difference (p > 0.05) in intracellular CE contents among different LSrHDLs-treated groups. 3.5.2. Intracellular Lipid Droplets Stained by Oil Red O. The intracellular lipid droplets of the cells treated with different LS-rHDLs at their respective Dm concentrations are displayed in Figure 7b. In comparison with normal cells (control group A (7.13 ± 3.28)), foam cells had the large red disposition as a positive control (group E (101.58 ± 3.94)) (Figure 7a). LS-

remained at relatively high levels when the concentrations of LS were less than 10 μM. Moreover, it also revealed that the rHDL carrier had little cytotoxicity on RAW264.7 cells at the concentrations from 0 to 800 μg/mL. 3.4. Evaluation of LS-rHDLs Based on the MedianEffect Principle. The inhibitory effects of the rHDL carrier and LS on DiI-oxLDL internalization in RAW264.7 cells were shown in Figure S1. To evaluate their combined effect, the dose−effect relationships were established first. Figure 4

Figure 4. Dose−effect curves of rHDL carrier (A) and LS (B) with the inhibitory effect on DiI-oxLDL internalization in RAW264.7 cells (n = 3).

illustrates the dose−effect curves of rHDL and LS. The Dm values of rHDL and LS calculated as the dose of drug required to produce the median effect were 273.82 and 1.70 μg/mL, respectively (Table 2). As shown in Table 2, the Dm values of Table 2. Dose−Effect Parameters of LS, rHDL Carrier, and Different LS-rHDLs with the Inhibitory Effect on DiI-oxLDL Internalization in RAW264.7 Cells (n = 3) parametersa preparations LS rHDL carrier ratio/ Dm,LS:Dm,rHDL LS3:1 rHDL 6:1 9:1

CI valueb

Dm (μg/ mL)

r

1.70 273.82

0.98 1.00

43.94 30.82 25.87

0.98 0.97 0.98

ED40

ED45

ED50

1.00 0.61 0.72

1.11 0.67 0.80

1.25 0.76 0.89

a

Dm is the median-effect dose (concentration that inhibits the uptake of DiI-oxLDL by 50%), r is the linear correlation coefficient of the median-effect plot. bCI is the combination index (CI < 1, synergism; CI = 1, additive effect; CI > 1, antagonism).

Figure 5. CI-fa curves of 3:1 (Dm,LS:Dm,rHDL) LS-rHDL (A), 6:1 (Dm,LS:Dm,rHDL) LS-rHDL (B), and 9:1 (Dm,LS:Dm,rHDL) LS-rHDL (C) in RAW264.7 cells (n = 3). 1022

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Figure 6. Intracellular cholesterol esters contents when the RAW 264.7 cells were treated with different LS-rHDLs at their respective Dm concentrations. Normal control group (A), 3:1 (Dm,LS:Dm,rHDL) LSrHDL (B), 6:1 (Dm,LS:Dm,rHDL) LS-rHDL (C), 9:1 (Dm,LS:Dm,rHDL) LSrHDL (D), and the positive control group (E). **, p < 0.01 (mean value ± SD, n = 3).

Figure 8. Cholesterol efflux capabilities of LS, LS-liposome, rHDL, and 6:1 (Dm,LS:Dm,rHDL) LS-rHDL in RAW 264.7 cells. *, p < 0.05; **, p < 0.01 (mean value ± SD, n = 3).

rHDLs-treated groups were all obtained at about 50% of lipid disposition with no significant difference (p > 0.05). 3.5.3. Cholesterol Efflux Assay. Since the LS-rHDL formulated with 6:1 (Dm,LS:Dm,rHDL) was proven to exhibit the best synergistic effect as demonstrated by the lowest CI values, RAW 264.7 cells were treated with this preparation in the assay of cholesterol efflux for the validation of synergism between rHDL and LS. As illustrated in Figure 8, the

cholesterol efflux capability was quite low in cells treated with free LS (5.53%), but a slight increment was found in the treatment with LS-liposome (8.07%). Compared with LSliposome, cholesterol efflux capability of the rHDL carrier (21.03%) was significantly increased (p < 0.01). Interestingly, a synergistic cholesterol efflux capacity (29.85%) was observed in the LS-rHDL (p < 0.05, compared LS-rHDL with rHDL; p < 0.01, compared LS-rHDL with LS).

Figure 7. Intracellular lipid deposition stained by oil red O when the RAW 264.7 cells were treated with different LS-rHDLs at their respective Dm concentrations. Normal control group (A), 3:1 (Dm,LS:Dm,rHDL) LS-rHDL (B), 6:1 (Dm,LS:Dm,rHDL) LS-rHDL (C), 9:1 (Dm,LS:Dm,rHDL) LS-rHDL (D), and positive control group (E). **, p < 0.01 (mean value ± SD, n = 3). 1023

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with acute coronary syndrome.35 Therefore, utilizing rHDL as a vehicle of cardiovascular drug may not only enhance the plaque targeting efficiency, but also improve the antiatherosclerotic effect. In this study, the mouse macrophage cell line RAW 264.7, a commonly used cell line to establish the foam cell model through modified LDL stimulation,36 was used in the evaluation of LS and rHDL. In order to identify the targeting efficiency of the rHDL carrier in macrophage cells, fluorescent dye coumarin-6 (C6) was loaded into rHDL. As illustrated in Figure 2, rHDL showed high targeting efficiency as compared to that of liposome, which might be attributed to the binding of apoA-I to the SR-BI receptor in macrophage cells.37 To further confirm the interaction of apoA-I with SR-BI in mediating the uptake of rHDL, the selective inhibitor of SR-BI and the excess amount of apoA-I were used to treat the cells, respectively. The decrement of rHDL uptake after pretreatment either with BLT1 or the excess apoA-I demonstrated that rHDL could target plaque macrophage cells through SR-BI-mediated internalization. The primary goal of the present research was to explore whether the rHDL carrier and the encapsulated lovastatin (LS) exert the synergistic effect. To address this issue, the LS-rHDL drug delivery system was regarded as a drug combination of LS and rHDL carrier. The median-effect analysis was employed for the evaluation of their combined effect. To investigate the combined effect of LS and rHDL, the dose−effect relationships were established first. With regard to the dose, the dose ranges of LS and rHDL were selected within their safe concentration ranges based on the results of in vitro cytotoxicity. The results demonstrated that the cells treated with various concentrations of LS ranging from 0 to 10 μM yielded relatively high viability rates. Moreover, high cell viabilities were obtained when the concentrations of rHDL were less than 800 μg/mL (Figure 3). In the pathogenesis of AS, inflammation has been involved in every stage of AS from endothelial cell dysfunction to plaque rupture.38 Internalization of oxLDL has been recognized as a requisite and the initiating event in the pathogenesis of atherosclerosis,39 which could trigger a cascade of inflammatory reactions. Hence, inhibition effect on oxLDL internalization was chosen as an index of drug efficacy. A great number of studies have demonstrated that LS and rHDL could inhibit the uptake of oxLDL by suppressing the expression of oxLDL uptake-related receptors.40−42 It is widely accepted that the lower Dm value (i.e., the dose of drug required to produce the median inhibitory effect) represents the stronger inhibitory effect. As shown in Table 2, compared with the rHDL carrier, LS showed a remarkable inhibitory effect on DiI-oxLDL internalization as demonstrated by the low Dm value (i.e., 161fold lower than the rHDL carrier). LS-rHDLs were then formulated with the varied LS/rHDL ratios based on their respective Dm values. To ensure the high drug entrapment efficiency and the favorable stability, the ratios of LS/rHDL were set at 3:1, 6:1, and 9:1 (Dm,LS:Dm,rHDL). In vitro characterizations were carried out to examine the physicochemical properties of LS-liposomes and LS-rHDLs. Table 1 shows that three different LS-rHDLs possessed the desirable EE. The increase in negative surface charge and the discoidal shape of LS-rHDL suggested the successful binding of apoA-I to the surface of the lipid core.43 The morphological characteristic of LS-rHDL (Figure 1) was identical with our previous studies.33

3.6. Synergistic Mechanism Study of rHDL and LS by Western Blotting. In order to understand the mechanism underlying the synergism between the rHDL carrier and LS, the expression of oxLDL uptake-related principal receptors (i.e., CD36 and SR-A) was investigated since the inhibitory effect on oxLDL internalization in macrophages was the principal drug effect index in the evaluation of the combined effect of LS and rHDL. The protein expression of SR-A or CD36 in this study was indicated with the SR-A or CD36 expression level relative to the expression of GAPDH. As depicted in Figure 9, LS exerted

Figure 9. Western blot analysis of CD36 and SR-A expression in RAW 264.7 cells. Normal control group (A), LS (B), rHDL (C), and 6:1 (Dm,LS:Dm,rHDL) LS-rHDL (D). *, p < 0.05; **, p < 0.01 (mean value ± SD, n = 3).

a stronger inhibitory effect on the expression of SR-A and CD36 than that of rHDL (p < 0.01). Compared with LS and rHDL, LS-rHDL displayed a potent suppression on SR-A expression (p < 0.01, compared LS-rHDL with rHDL; p < 0.05, compared LS-rHDL with LS). However, a slight upregulation of CD36 expression was observed in LS-rHDL as compared to LS.

4. DISCUSSION Atherosclerosis (AS), a progressive chronic inflammatory disease characterized by the accumulation of cholesterol and lipid within the artery walls, is the leading cause of mortality in Western countries.34 Several lines of evidence have shown that rHDL exhibited the atherosclerotic plaque targeting effect, owing to the interaction of apoA-I with the overexpressed SRBI receptor in plaque macrophages and foam cells.3,4 In our previous study, an ex vivo imaging study demonstrated that rHDL could bind easily to atherosclerotic lesions,1,5 which further confirmed the plaque targeting efficiency of rHDL. Furthermore, increasing evidence have been demonstrated that administration of rHDL in atherosclerotic animal models yielded favorable results.2 The results of clinical trials have also proven that rHDL could regress the atheroma of patients 1024

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expression. However, the upregulation of CD36 expression was observed in LS-rHDL. This may be interpreted by the reciprocal upregulated effect between CD36 and SR-A.50

Since the high cell viability rates were obtained when the cells were treated with different LS-rHDLs at the concentration of 300 μg/mL (Figure S2), the dose ranges of different LSrHDLs were all selected within the concentrations from 0 to 300 μg/mL. It was revealed that the rHDL carrier and LS were synergistic at low effect levels and antagonistic at high effect levels. Based on the lowest CI values, the best synergistic ratio of LS/rHDL was determined at 6:1 (Dm,LS:Dm,rHDL) (Figure 5). For verifying the accuracy of the results obtained from the median-effect analysis, different drug effect indexes were employed in the study. Besides the inhibition effect on uptake of oxLDL, the content of intracellular lipid droplets could be selected as another index of drug effect since oxLDL internalization is positively correlated with the amount of lipid droplets.44 In this study, the lipid disposition of cells was analyzed by a cholesterol quantification assay and oil red O staining, respectively. Fifty percent of reduction on the intracellular lipid contents was desired to obtain when the cells were treated with different LS-rHDLs at their respective Dm concentrations. As expected, the results of the cholesterol quantification assay manifested that about 50% of CE contents was reduced when cells were treated with LS-rHDLs at their respective Dm concentrations (Figure 6), suggesting that the Dm values acquired from the median-effect analysis were accurate. A similar conclusion was obtained from the qualitative analysis by oil red O staining (Figure 7). In order to further validate the synergistic effect of the rHDL carrier and LS, cholesterol efflux capability of LS-rHDL was measured. The results demonstrated that LS had little effect on cholesterol efflux, but a slight increment in mediating cholesterol efflux was found when LS was incorporated into the liposome, which might be associated with the more efficient internalization of LS in the treatment with LS-liposome than that of the free drug.27 Moreover, the bilayer membrane of liposome may also be responsible for it since it could receive cholesterol.45 However, the increase of cholesterol efflux was limited owing to the inefficient passive diffusion driven by the large cholesterol concentration gradient between cell and medium.29 Compared with the LS-liposome, the cholesterol efflux rate of the rHDL carrier was significantly increased, which may be interpreted by the more efficient active cholesterol efflux pathway mediated by apoA-I through the interaction with efflux membrane proteins, such as SR-BI, ABCA-1, and ABCG-129,46,47 (p < 0.01). Interestingly, it was found that LS-rHDL exhibited a synergistic cholesterol efflux capacity. It might be ascribed to the combined effect of the passive and active efflux pathway. Collectively, the median-effect analysis suggested that the rHDL carrier and LS exhibited a synergistic anti-atherosclerotic effect at the low effect levels. However, the mechanism of synergy between rHDL and LS was unclear. Macrophages have been shown to express several scavenger receptors that are responsible for the internalization of oxLDL, including SR-A, CD36, CD68, and LOX-1, etc..48 Among them, SR-A and CD36 have been considered to be the principal receptors to bind and internalize oxLDL, which accounted for most of the internalization of oxLDL (c.a. 75−90%).49 Therefore, we hypothesized that the expression of these receptors might be related to the synergistic effect of rHDL and LS. Therefore, to gain a better understanding of the synergistic mechanism between rHDL and LS, the suppression effects on CD36 and SR-A expression were investigated. The results manifested that LS-rHDL displayed a synergistic suppression on SR-A

5. CONCLUSIONS In this study, we investigated the combined effect of the rHDL carrier and the encapsulated LS based on the median-effect principle. It was found that rHDL and LS were synergistic in inhibiting the internalization of DiI-oxLDL at the low effect levels. It could produce a desirable synergism when LS-rHDL was formulated with LS/rHDL at a ratio of 6:1 (Dm,LS:Dm,rHDL), which was further confirmed by the assay of cholesterol efflux. The synergism mechanism between LS and the rHDL carrier might be interpreted by their inhibitory effects on SR-A expression. We believed that median-effect analysis could be utilized to investigate the dose−effect relationships of the biofunctional carrier and the encapsulated drug, which might provide guidance to the design of biofunctional drug delivery systems. Further in vivo studies are required to validate the synergism of rHDL and LS.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00923. The inhibitory effect of rHDL and LS on DiI-oxLDL internalization, in vitro cytotoxicities LS-rHDLs, and the DRI values of LS-rHDLs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]; Tel: +86-25-83271293; Fax: +86-25-83271293. ORCID

Jianping Liu: 0000-0003-1825-7122 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 81773669), National Science and Technology Major Project (No. 2017YFA0205400), and the Postgraduate Innovation Funds of Huahai Pharmaceutical (No. CX16B-002HH). The research was also supported by the Graduate Cultivation Innovative Project of Jiangsu Province (No. KYCX17_0677) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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ABBREVIATIONS HDL, high-density lipoprotein; rHDL, recombinant highdensity lipoprotein; AS, atherosclerosis; RCT, reverse cholesterol transport; apoA-I, apolipoprotein AI; SR-BI, scavenger receptor BI; ABCA-1, ATP-binding cassette transporter A1; ABCG-1, ATP-binding cassette transporter G1; LS, lovastatin; HMG-CoA, 3-hydroxy-3-methylglutaryl−coenzyme A; LS-liposome, lovastatin-loaded liposome; LS-rHDL, lovastatin-loaded recombinant high-density lipoprotein; LDL, low density lipoprotein; ox-LDL, oxidized low density lipoprotein; CI, 1025

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combination index; Dm, median-effect dose; EE, drug entrapment efficiency; DL, drug loading efficiency; TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester; TEM, transmission electron microscopy; C6, coumarin-6

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