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Evaluation of the Combined Effect of Recombinant HighDensity Lipoprotein Carrier and the Encapsulated Lovastatin in RAW264.7 Macrophage Cells Based on the Median-effect Principle Cuiping Jiang, Yi Zhao, Yun Yang, Jian Hua He, Wenli Zhang, and Jianping Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00923 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018
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Molecular Pharmaceutics
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Evaluation of the Combined Effect of Recombinant High-Density Lipoprotein
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Carrier and the Encapsulated Lovastatin in RAW264.7 Macrophage Cells Based on
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the Median-effect Principle
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Cuiping Jiang, Yi Zhao, Yun Yang, Jianhua He, Wenli Zhang*, Jianping Liu*
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Department of Pharmaceutics, China Pharmaceutical University, Nanjing, PR China
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Corresponding author: Professor Jianping Liu
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E-mail:
[email protected] 8
No. 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
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Tel: +86-25-83271293; Fax: +86-25-83271293.
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Co-corresponding author： ：Associate professor Wenli Zhang
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E-mail:
[email protected] 12
No. 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
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For Table of Contents Use Only
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Title: Evaluation of the Combined Effect of Recombinant High-Density Lipoprotein
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Carrier and the Encapsulated Lovastatin in RAW264.7 Macrophage Cells Based on the
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Median-effect Principle
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Authors: Cuiping Jiang, Yi Zhao, Yun Yang, Jianhua He, Wenli Zhang*, Jianping Liu*
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Molecular Pharmaceutics
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Abstract
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Recombinant high-density lipoprotein (rHDL) displays similar anti-atherosclerotic
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effect with native HDL and could also be served as a carrier of cardiovascular drug for
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atherosclerotic plaque targeting. In our previous studies, rHDL has shown more potent
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anti-atherosclerotic efficacy as compared to other conventional nanoparticles with a
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payload
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anti-atherosclerotic effect of rHDL carrier and the encapsulated LS might exist. In this
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study, the dose-effect relationships and combined effect of rHDL and LS were
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quantitatively evaluated in RAW 264.7 macrophage cells using the median-effect analysis,
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in which rHDL carrier was regarded as a drug combined. Median-effect analysis
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suggested that rHDL and LS exerted a desirable synergistic inhibition on the oxLDL
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internalization at a ratio of 6:1 (Dm LS: Dm rHDL) in RAW 264.7 macrophage cells. About
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50% of reduction on intracellular lipid contents was found when RAW264.7 cells were
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treated with LS-loaded rHDLs at their respective median-effect dose (Dm) concentrations
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and a synergistic effect on mediating cholesterol efflux was also observed, which verified
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the accuracy of the results obtained from the median-effect analysis. The mechanism
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underlying the synergistic effect of rHDL carrier and drug might be attributed to their
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potent inhibitory effects on SR-A expression. In conclusion, median-effect analysis was
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proved to be a feasible method to quantitatively evaluate the synergistic effect of
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bio-functional carrier and the drug encapsulated.
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Keywords: recombinant high-density lipoprotein, median-effect principle, synergistic
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effect, drug combination, atherosclerotic targeting
of
lovastatin
(LS).
Therefore,
we
hypothesized
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that
a
synergistic
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Abbreviations
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HDL
high-density lipoprotein
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rHDL
recombinant high-density lipoprotein
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AS
atherosclerosis
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RCT
reverse cholesterol transport
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apoA-I
apolipoprotein AI
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SR-BI
scavenger receptor BI
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ABCA-1
ATP-binding cassette transporter A1
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ABCG-1
ATP-binding cassette transporter G1
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LS
lovastatin
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HMG-CoA
3-hydroxy-3-methylglutaryl–coenzyme A
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LS-liposome
lovastatin-loaded liposome
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LS-rHDL
lovastatin-loaded recombinant high-density lipoprotein
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LDL
low density lipoprotein
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ox-LDL
oxidized low density lipoprotein
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CI
combination index
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Dm
median-effect dose
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EE
drug entrapment efficiency
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DL
drug loading efficiency
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TC
total cholesterol
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FC
free cholesterol
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CE
cholesterol ester
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TEM
transmission electron microscopy
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C6
coumarin-6
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Molecular Pharmaceutics
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1. Introduction
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High-density lipoprotein (HDL) is an important plasma lipoprotein in lipid transport
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system 1, whose level is considered to be inversely correlated with the incidence of
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atherosclerosis (AS) 2. The anti-atherogenic activities of HDL are mainly attributed to
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reverse cholesterol transport (RCT) 3, during which HDL is capable of removing
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excessive cholesterol from plaque macrophages and foam cells to liver for biliary
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excretion through interaction of apoA-I with scavenger receptor BI (SR-BI),
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ATP-binding cassette transporter A1 (ABCA-1) and G1 (ABCG-1) receptors 4. Along
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with RCT, HDL possesses a multitude of other cardiovascular protective effects,
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including the anti-oxidative effect, the endothelial protective function as well as the
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anti-thrombotic property 5.
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Several lines of evidences have shown that recombinant HDL (rHDL) possesses 6-7
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atheroprotective effect similar to the native HDL
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increasing attention as a drug vehicle due to the favorable attributes, including lipid space
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for hydrophobic drugs, long circulation time in blood and the capacity to evade
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reticuloendothelial system, etc. 8-10. Besides, rHDL could target the atherosclerotic plaque
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via the over-expressed SR-BI receptor in plaque macrophages and foam cells
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Therefore, rHDL may serve as a bio-functional drug delivery candidate of cardiovascular
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drugs.
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. Recently, rHDL has gained
4, 11
.
Lovastatin (LS), a member of the 3-hydroxy-3-methylglutaryl–coenzyme A 12-13
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(HMG-CoA) reductase inhibitors, is widely used as an anti-atherosclerotic drug
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Besides blood lipid lowering effect, LS plays an important role in attenuating vascular 14
.
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plaque inflammation
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endothelial dysfunction, oxidative stress and thrombosis 15. In our previous studies, rHDL
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carrier has been utilized with a payload of LS for the improved anti-atherosclerotic
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therapy
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plaque targeting efficiency and more potent anti-atherogenic efficacy in atherosclerotic
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rabbit model compared with the other LS-loaded nano drug delivery systems
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Therefore, a synergistic anti-atherosclerotic effect of LS and rHDL carrier may exist. As
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such, investigating their dose-effect relationships would be beneficial for the evaluation
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of their combined effect.
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, which is ascribed to an interplay of pleiotropic effects on
. The results demonstrated that LS-loaded rHDLs (LS-rHDLs) had higher
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.
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Median-effect principle, based on the mass-action law, is a general theory of
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dose-effect relationship 18. Combination index (CI) derived from median-effect equation 5
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is an effective scientific index to depict the combined effect of two or multiple
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compounds, where CI >1, =1 and
