Preventive effects of simvastatin nanoliposome on isoproterenol-induced cardiac remodeling in mice

    Preventive Effects of Simvastatin Nanoliposome on Isoproterenol-Induced Cardiac Remodeling in Mice Nuerbiye Tuerdi, Lu Xu, Baoling Zhu, Cong Chen, Yini Cao, Yunan Wang, Qiang Zhang, Zijian Li, Rong Qi PII: DOI: Reference:

S1549-9634(16)30049-1 doi: 10.1016/j.nano.2016.05.002 NANO 1338

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

14 January 2016 16 April 2016 1 May 2016

Please cite this article as: Tuerdi Nuerbiye, Xu Lu, Zhu Baoling, Chen Cong, Cao Yini, Wang Yunan, Zhang Qiang, Li Zijian, Qi Rong, Preventive Effects of Simvastatin Nanoliposome on Isoproterenol-Induced Cardiac Remodeling in Mice, Nanomedicine: Nanotechnology, Biology, and Medicine (2016), doi: 10.1016/j.nano.2016.05.002

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ACCEPTED MANUSCRIPT Preventive Effects of Simvastatin Nanoliposome on IsoproterenolInduced Cardiac Remodeling in Mice

Zijian Li d, *, Rong Qi a, b, c, *

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Nuerbiye•Tuerdi #, a, b, c, Lu Xu #, a, b, Baoling Zhu #, d, Cong Chen a, b, Yini Cao a, b, Yunan Wang a, b, Qiang Zhang e,

a Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, Peking

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University, Beijing 100191, China

b Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, China

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c School of Basic Medical Science, Shihezi University, Shihezi 832000, Xinjiang, China d Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular

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Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China

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e School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China

# These three authors contribute equally to the paper.

Rong Qi, PhD

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Associate Professor

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* Corresponding authors:

Address: 38 Xueyuan Road, Haidian District, Peking University Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing 100191, China. E-mail: [email protected]; Tel: +86 10 8280 5164; Fax: +86 10 8280 5164.

Zijian Li, PhD Professor Address: 38 Xueyuan Road, Haidian District, Institute of Vascular Medicine, Peking University Third Hospital, Beijing 100191, China. E-mail: [email protected]; Tel: +86 10 8226 5519.

The study was supported by the grants from National Natural Science Foundation of China (no. 81270368, 81360054) and the National Basic Research Program of China (2015CB932100). We have no conflicts of interest to declare.

Word count for the abstract: 137, a complete manuscript (to include body text and figure legends): 5536. Number of references: 44; Figures: 6; Tables: 2; Supplemental Figures: 3; Supplemental Table: 1.

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ACCEPTED MANUSCRIPT Abstract In this study, simvastatin (SMV) and SMV nanoliposome (SMV-Lipo) were given to male

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BALB/c mice either by intragastric (i.g.) or intraperitoneal (i.p.) administration, and their effects on isoproterenol (ISO) induced cardiac remodeling were compared. The results indicate that by i.p. administration, the SMV-Lipo at an equal SMV dose exhibited more

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significant inhibitive effects than the crude SMV on cardiac hypertrophy, fibrosis and inflammation. Comparing the SMV-Lipo on different administration regimens, i.p. group

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showed more significant inhibitive effects on cardiac remodeling than i.g. group. In addition,

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pharmacokinetic studies revealed that SMV-Lipo administrated by either i.p. or i.g. more significantly improved the plasma SMV concentration than the crude SMV. Therefore, the SMV-

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Lipo significantly enhanced the inhibitive effects of SMV on cardiac remodeling resulted from the enhanced absorption of SMV by nanoliposome formulation, and i.p. was better than i.g.

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administration.

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Key words: Simvastatin; nanoliposome; isoproterenol; cardiac remodeling; bioavailability

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ACCEPTED MANUSCRIPT Background Cardiac remodeling is an adaptive response to pathophysiological stresses, including

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hypertension-associated pressure overload and ischemia-reperfusion injury after myocardial infarction, which can progress to pathological remodeling, arrhythmia and heart failure in the longer term(1). At the level of cardiomyocyte, myocardium remodeling is characterized by

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increased cell hypertrophy, aggravated necrotic cell death(2), and reactivation of the fetal cardiac genes. Concurrently, the phenotype of cardiac fibroblasts (CF) become myofibroblasts,

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which enhance collagen deposition (fibrosis)(3). Isoproterenol (ISO), a nonselective β-

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adrenergic receptor (β-AR) agonist, is associated with severe myocardial hypertrophy and injury(4, 5). It has been reported that excessive β-AR stimulation induces cardiac remodeling

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associated with hypertrophy and fibrosis as well as cardiomyocyte apoptosis both in vivo and in vitro(6, 7).

Simvastatin (SMV), an inhibitor of 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase, is a

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widely used anti-hypercholesterolemia drug(8). However, beneficial effects of SMV are more than lowering cholesterol alone(9-12). Previous studies have clearly revealed cardiac

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protection effects of SMV(13, 14), including improving endothelial function, reducing oxidative stress, stabilizing atherosclerotic plaques and reducing vascular inflammation. Several studies have reported that high-dose statin therapy can further reduce cardiovascular risk compared with standard regimens(15, 16). In addition, statins have also been found to protect against cardiac remodeling both in vivo and in vitro(17, 18). However, adverse effects of statins have increasingly aroused people's attention(19-22). The main myotoxicity of SMV is myalgia and rhabdomyolysis(23, 24). These side effects can be attenuated by decreasing the dosage or terminating the therapy of statin. Moreover, SMV is a water insoluble drug and has irregular intestinal absorption, which influences its absorption and results in a low oral bioavailability of less than 5%(25). Overall, the side effects and low bioavailability result in 3

ACCEPTED MANUSCRIPT unsatisfied therapeutic effects of SMV. Therefore, if an advanced drug delivery system can be used to improve the solubility and absorption of SMV, its dosage could be lowered and maximal therapeutic effects could be achieved with minimal side effects.

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Nanoliposome have long been recognized as a useful dosage form for increasing drug absorption in circulation through encapsulating hydrophobic drugs within their lipid bilayers

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to increase solubility and stability of the loaded drugs. In addition, the liposomal bilayer mimics the structure of cell membranes and improves intracellular delivery efficacy of the

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encapsulated drugs by fusing with cell membranes(26). Our previous study indicates that SMV

bioavailability of SMV(27).

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nanoliposome significantly improved the solubility, transepithelial transport and oral

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In this study, SMV nanoliposome (SMV-Lipo) were prepared, which significantly enhanced the inhibitory effects of SMV on ISO-induced cardiac remodeling. In addition, plasma SMV concentrations of either SMV or SMV-Lipo treated mice by intraperitoneal (i.p.) or gavage (i.g.)

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administration were determined to explain the improvement of SMV absorption and

Methods Materials

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pharmacodynamics effects by the nanoliposome formulation.

Isoproterenol was purchased from Sigma (Beijing, China). SMV (≥98%) was purchased from Haizheng Co., Ltd. (Zhejiang, China). Isoflurane was purchased from Baxter Healthcare Corporation (New Providence, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). 5 × All-In-One RT Master Mix and EvaGreen 2 × qPCR Master Mix were purchased from Applied Biological Materials Inc. (British Columbia, Canada). Mac-2 antibody was purchased from Abcam (Cambrige, UK). Methanol (Chromatography Grade) was purchased from Xihua Special Reagent Factory (Tianjin, China). Acetonitrile (Chromatography Grade) was purchased 4

ACCEPTED MANUSCRIPT from Merck (Germany). All the other reagents were provided by local suppliers, unless otherwise mentioned.

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Preparation and characterizations of the SMV-Lipo The SMV-Lipo was prepared by using a thin film dispersion method(28). To prepare lipid films, soybean lecithin (225 mg), cholesterol (25 mg) and SMV (25 mg) were mixed in a ratio

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of 9:1:1 and dissolved in 3 mL chloroform. The solvent was slowly evaporated in a rotary

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evaporator at 37 °C for 30 min to achieve a lipid film, then dried overnight under vacuum and at room temperature. Multilamellar vesicles (MLVs) were prepared by hydrating the lipid film

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with 6 mL PBS (pH 7.5), and the resultant suspensions were sonicated for 30 min using a probe sonicator to obtain small unilamellar vesicles (SUVs) of SMV-Lipo. Size, zeta-potential

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and PDI of the SMV-Lipo were measured by Malvern Zetasizer Nano-ZS (Santa Barbara, CA, USA). Drug encapsulation efficiency (EE) in the SMV-Lipo was calculated by a formula as follows:

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EE % = (Mtotal- Mfree) / Mtotal × 100%

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Mtotal and Mfree were the mass of total and free SMV in the nanoliposome system, respectively. The drug loading percentage in nanoliposomes was calculated as follows: Drug loading % = Mencapsulated drug/Mcarriers × 100% Mencapsulated drug was the mass of SMV encapsulated in the nanoliposomes, Mcarrier was the mass of the lipids in the nanoliposome system. The amount of SMV was measured using High Performance Liquid Chromatography (HPLC, Shimadzu LC-15C, Japan) at a wavelength of 238 nm with a Purospher STAR C18 column (250 mm×4.6 mm, 5 μm) and acetonitrile/water (55/45) as mobile phase. Three repeated experiments were conducted for each sample. 5

ACCEPTED MANUSCRIPT Animal model All study protocols conformed to the Animal Management Rules of China (Documentation No.

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55, 2001, Ministry of Health, China). All experiments were approved by the Committee for Ethics of Animal Experiments and were conducted in accordance with the Guidelines for Animal Experiments, Peking University Health Science Center. All mice were raised under a

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12-hour light/dark cycle with free access to food and water.

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To determine the formulation effects of the nanoliposome on pharmacologic effect of SMV, 48 male BALB/c mice at an age of 10 weeks, weighing from 23 to 25g, were purchased from the

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Animal Department of the Peking University Health Science Center and randomly assigned to eight groups. The groups and drug administration regimens are shown in Table 1. Briefly,

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cardiac remodeling of the mice was induced by daily subcutaneous (s.c.) injection of ISO (5 mg/kg/d) for 7 days. For the ISO-induced and drug-treated groups, the mice received i.p. injection of SMV or SMV-Lipo at an SMV dose of 10 mg/kg/d, or i.g. administration of the SMV

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or SMV-Lipo at an SMV dose of 20 mg/kg/d began on the same day when ISO inducement started and lasted for 7 days. The mice received daily s.c. injection of saline and treated with

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saline by i.p. injection or i.g. administration were set as the control groups. Parallel experiments were done on no ISO-induced normal mice to investigate the effects of the SMV and SMV-Lipo on cardiac function and morphology of non-injured mice heart tissues. The groups and drug administration regimens are shown in Supplemental Table 1. Briefly, all mice received daily s.c. injection of the equal volume of saline instead of ISO and meanwhile treated with saline, the SMV or the SMV-Lipo by i.p. injection (10 mg/kg/d) or i.g. administration (20 mg/kg/d) and lasted for 7 days. After 7 days, cardiac function of the mice was assessed by echocardiography before the mice were sacrificed. Cardiac remodeling was evaluated by determination of the ratio of heart to body weight (HW/BW), as well as the ratio of heart weight to length of tibia (HW/TI). 6

ACCEPTED MANUSCRIPT Additionally, hematoxylin & eosin (HE) staining and picric sirius red staining were performed to evaluate morphology and fibrosis degree of the mice hearts, respectively. Real-time PCR was performed to detect expressions of the involved genes.

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Echocardiography and evaluation of left ventricular hemodynamics Mice were anesthetized with isoflurane (1% in 100% of oxygen). Echocardiographic images

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were obtained using a VisualSonicsVevo 770 High Resolution Imaging System (VisualSonics

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Incorporated, Toronto, Canada). Two-dimensional parasternal long-axis views and short-axis views were obtained at the level of the papillary muscle. Diastolic left ventricular posterior

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wall thickness (LVPW, d), systolic left ventricular posterior wall thickness (LVPW, s), left ventricular internal diameter at end-systole (LVID, s) and left ventricular internal diameter at

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end-diastole (LVID, d) were measured. Ejection fraction (EF%) and fraction shortening (FS%) were calculated from these parameters. All measurements were averaged from three consecutive cardiac cycles.

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HW/BW Ratio and HW/TI Ratio

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After sacrificing the mice, their hearts were perfused with cold phosphate-buffered saline (PBS) and weighed. The length of tibia was measured using a venire caliper 530-101 (SanFeng, Ningbo, China). The cardiac index was evaluated by the ratio of heart weight to length of tibia (HW/TI, mg/mm) and the ratio of heart weight to body weight (HW/BW, mg/g).

Morphology Hearts were harvested after sacrifice and perfused in retrograde with cold PBS, fixed with 4% paraformaldehyde overnight, then changed to 20% sucrose (m/v, dissolved with PBS) overnight, and embedded in paraffin. LV sections cut from the same location of each heart, and serial sections (6 μm thick) were stained with HE staining, or picric sirius red staining to 7

ACCEPTED MANUSCRIPT evaluate cardiomyocyte cross-sectional area and cardiac fibrosis. Cardiac collagen volume fraction was calculated as the ratio of the stained fibrotic area to total myocardial area by Image Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, USA). The same part of each heart was

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used for quantitative real-time PCR. For morphometrical analysis, images of left ventricle sections were observed using the Leica Q550 IW imaging workstation (Leica Microsystems

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Imaging Solutions Ltd., Cambridge, UK).

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Immunohistochemical staining

Mac-2 (a macrophage marker) was stained to detect macrophages in the heart sections by

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immunohistochemical method. Briefly, sections treated with 5% normal goat serum for 30 min were incubated with 200-fold diluted polyclonal primary antibodies (Santa Cruz, USA) for

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1 h following washing with PBS, after which the sections were incubated with 500-fold diluted goat-anti-rabbit IgG antibodies. The sections were then washed with PBS and incubated with DAB solution. The reaction was terminated with deionized water. Counterstaining with

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hematoxylin was performed after the sections were washed with PBS. The sections were immediately immersed in graded alcohol and xylene, and sealed with neutral gum. The

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prepared sections were observed under a light microscope (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK).

Real-time quantitative PCR Total RNA from each tissue was isolated according to the manufacturer’s instructions. cDNA was generated by reverse transcription of 5 µg of RNA using 5× All-In-One RT Master Mix. Real-time quantitative PCR was performed using EvaGreen 2 × qPCR Master Mix on a RealTime PCR (Stratagene Mx3000P Multiplex Quantitative PCR System, Agilent Technologies Inc., Santa Clara, CA, USA) with the following primers: for ANF, 5’- GCTTCCAGGCCATATTGGAG 3’(forward), 5’- GGGGGCATGACCTCATCTT -3’(reverse); for BNP, 5’- AGGACCAAGGCCTCACAA 8

ACCEPTED MANUSCRIPT AA -3’(forward),

5’-

ACTTCAAAGGTGGTCCCAGAG

-3’(reverse);

for Collagen-III,

5’-

CTGTAACATGGAAACTGGGGAAA -3’(forward), 5’- CCATAGCTGAACTGAAAACCACC -3’(reverse); for β-actin, 5’- GAGACGGAGCTGTTGGTAAAA -3’(forward), 5’- TCTTGCTCAGTGTCCTTGCTGG -

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3’(reverse). The cycling protocol entailed incubation at 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Relative mRNA expression levels were normalized to those of

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the housekeeping gene of β-actin.

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Plasma SMV concentrations

Blood samples from each group were collected and centrifuged (Dragon Lab DM1424, Beijing,

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China) at 1672 g and 4 °C for 10 min to separate plasma. Then SMV in the plasma samples was extracted by a direct precipitation method. Briefly, 150 μL dichloromethane and 450 μL

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cyclohexane were added into 50 μL plasma and the mixture was vortexed for 3 min. The mixture was then centrifuged at 1672 g for 15 min. The supernatant was isolated and dried by nitrogen gas blowing in a 40 °C water bath. The remnant was dissolved with 100 μL mobile

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phase by vortexing for 3 min, and then centrifuging at 10450 g for 10 min. SMV concentrations in the supernatant were determined by HPLC.

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HPLC chromatographic conditions for analysis of SMV concentrations included a Purospher STAR C18 column (250 mm × 4.6 mm, 5 µm) and a mobile phase of acetonitrile and water at a ratio of 55/45 with a flow rate of 1.0 mL/min. The detection wavelength was 238 nm and injection volume of the samples was 20 μL.

Statistical analysis All data are presented as mean ± SD. Statistical analysis was performed using unpaired twotailed t-test by SPSS 19.0. P＜0.05 was considered statistically significant.

Results

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ACCEPTED MANUSCRIPT Characterizations of the SMV-Lipo Particle size, PDI, zeta potential and encapsulation efficiency of the SMV-Lipo are shown in Table 2. The results indicate that the particle size of the SMV-Lipo was 121.4 ± 5.5 nm. The

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encapsulation efficacy of SMV in the nanoliposomes was 95.98±1.35% and SMV loading percentage (encapsulated mass/carrier mass, g/g) was 7.33±0.62%.

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Effects of the SMV-Lipo on left ventricular hypertrophy

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The ISO group (model group) exhibited significant increase in the HW/BW ratio as well as HW/TI ratio (Figure 1), indicating that cardiac hypertrophy had been established in the model

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group. When given at the same dosage and administration regimen, the SMV-Lipo significantly declined the HW/TI ratio. Notably, even when i.p. dose was half of the i.g. dose, the SMV-Lipo

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i.p. showed remarkable inhibitive effect on HW/TI ratio (P<0.01). Besides, SMV alone only had significant inhibition on the ratio of HW/TI at i.p. but not i.g. administration even the dosage of i.g. was doubled. The results indicate that the SMV-Lipo declined the ratios of HW/BW and

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HW/TI more significantly and i.p. administration was more efficient than i.g. administration.

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The results from the no ISO-induced mice indicate that there was no significant difference among the control, SMV and SMV-Lipo groups in the HW/BW ratio as well as HW/TI ratio (Supplemental Figure 1), which suggests that the SMV and SMV-Lipo had no influence in the normal mice hearts. As there was no significant difference between the i.p. and i.g. administration of saline in the control groups or the model groups, only one set of representative data were shown in all of the results.

Effects of the SMV-Lipo on cardiac functions Echocardiography revealed that cardiac hypertrophy was successfully established in the model group with a significant increase in diastolic left ventricular posterior wall thickness (LVPW, d) compared to the control group (0.61 ± 0.03 vs 0.89 ± 0.09, P<0.001) and this 10

ACCEPTED MANUSCRIPT parameter was significantly decreased both in the SMV group and the SMV-Lipo group (P<0.05) at i.p. but not i.g. administration (Figure 2A and 2B). The results of EF%, FS%, left ventricular internal diameter at end-systole (LVID, s) and left ventricular internal diameter at

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end-diastole (LVID, d) showed that there was no significant difference among the SMV group, SMV-Lipo group and the model group (Figure 2C to 2F), which indicate that both of the SMV

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and SMV-Lipo would not affect cardiac function on this model.

Similarly, echocardiography results from the no ISO-induced mice elucidated that neither the

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SMV nor SMV-Lipo would affect left ventricular structure (Supplemental Figure 2A & 2B) and

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cardiac function (Supplemental Figure 2C to 2F) of the non-injured mice hearts.

Effects of the SMV-Lipo on expression of the fetal cardiac genes

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The results of real-time PCR in Figure 3 showed that relative expressions of mRNA of ANF and BNP were elevated significantly in the ISO-induced group, indicating the reactivation of the fetal cardiac genes. In both i.p. and i.g. administration groups, the expression of ANF and BNP

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were significantly down-regulated in the SMV-Lipo groups (P<0.05), but not in the SMV groups. Additionally, the SMV-Lipo given by either i.p. or i.g. administration showed

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comparably inhibitive effects, even the dose of i.p. administration was half than that of the i.g. administration.

Effects of the SMV-Lipo on cardiac fibrosis The picric sirius red staining showed that the mice in the model group displayed severe cardiac interstitial fibrosis compared with those mice in the control group (Figure 4A). Quantitative analysis revealed that compared to the control group, the cardiac interstitial fibrosis area was significantly increased in the model group (Figure 4B). Consistent with this result, quantitative RT-PCR analysis demonstrates that the relative mRNA content of myocardial collagen-III was significantly increased in the model group (Figure 4C). The picric 11

ACCEPTED MANUSCRIPT sirius red staining and quantitative analysis revealed that by either i.p. or i.g. administration, the SMV and SMV-Lipo alleviated cardiac fibrosis to different extent. It was notable that the SMV-Lipo more greatly inhibited cardiac fibrosis compared with the SMV, and expression of

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collagen-III was down-regulated more significantly by the SMV-Lipo than the SMV both at i.p. and i.g. administration (P<0.01), and i.p. administration was more efficient than i.g.

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administration.

The results of HE (Supplemental Figure 3A) and picric sirius red staining (Supplemental

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Figure 3B) from the no ISO-induced mice further reinforce that both of the SMV and SMV-Lipo

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would not change the morphology and extra cellular matrix of the non-injured normal mice hearts.

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Effects of the SMV-Lipo on inflammation

HE staining showed cardiac interstitial infiltration of numerous inflammatory cells in the model group (Figure 5A). The immunohistochemical staining with Mac-2 antibody clearly

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showed that the mice from the ISO model group exhibited severe infiltration of macrophages

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(Figure 5B). At i.p. administration, the SMV slightly repressed the infiltration of macrophages, but the SMV-Lipo obviously decreased macrophage infiltration, which indicates that the SMVLipo had advantages over the SMV on anti-inflammation effect. The same tendency can be seen in the i.g. administration groups, but was less effective than the i.p. administration groups. Relative mRNA expression of F4/80 in Figure 5C further reinforces the results of immunohistochemical staining. The elevated expression of F4/80 induced by ISO was significantly declined in the SMV-Lipo groups both at i.p. and i.g. administration (P<0.05), but was not in the SMV groups (P＞0.05).

Plasma concentrations of SMV The results showed that the plasma SMV concentration was significantly higher in the SMV12

ACCEPTED MANUSCRIPT Lipo group than in the SMV group at the same dosage and administration regimen (Figure 6). Notably, when evaluating the plasma concentrations of SMV at different administration, i.p. administration of the SMV given at the half dosage of i.g. showed significantly higher plasma

was more efficient than i.g. administration of the SMV.

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Discussion

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concentration than that of i.g. administration of the SMV, suggesting that i.p. administration

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In our previous work, we demonstrate that the SMV-Lipo significantly improved the oral bioavailability of SMV, which was attributed to the great solubilization and highly efficient

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clathrin-mediated transepithelial transport of the nanoliposomal formulation to SMV(27). Based on this, in the current study we hypothesized that the preventive effects of SMV on ISO-

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induced cardiac remodeling, a well-proved pharmacologic effect of SMV beyond its lipidlowering effect(29-31), would be enhanced by the nanoliposome formulation. We prepared the SMV-Lipo by using the same thin film dispersion method(28) as we reported in the

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previous work and resulted in comparable SMV encapsulation efficiency and particle size of the SMV-Lipo. The results of stable plasma SMV concentrations revealed that in consistent

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with our previous results, i.g. administration of the SMV-Lipo to mice significantly improved the plasma concentration of SMV than the crude SMV, indicating better absorption as well as greater pharmacologic effects of the SMV-Lipo. In addition, comparing with i.g., i.p. administration of the SMV at a half dose of i.g. had significantly higher stable plasma SMV concentration than i.g. administration of the SMV, indicating the absorption of the SMV was more efficient at i.p. than i.g. administration. In comparison with the SMV, the nanoliposome formulation significantly improved oral absorption of SMV, thus resulted in the comparably high plasma SMV concentrations after i.g. or i.p. administration of the SMV-Lipo. Cardiac hypertrophy is a main compensatory response resulted from the increased afterload, acute myocardial injury or infection, with increased size of cardiomyocytes and number of 13

ACCEPTED MANUSCRIPT myofibroblasts(1, 32). The unmatched neogenesis of organelles and vessels cannot supply enough energy, which eventually leads to necrosis of cardiomyocytes and loss of contractile function of the heart. In this study, both of the SMV and SMV-Lipo administrated by either i.p.

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or i.g. for 7 days at present dosages were proved to have no impact on the non-injured normal mice hearts in terms of cardiac structure, function and morphology. The SMV and the SMVLipo administrated by i.p. injection significantly inhibited cardiac hypertrophy induced by ISO.

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However, for i.g. administration, only the SMV-Lipo group significantly inhibited the ratio of

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HW/TI as there was no difference between the SMV and the model group. It was reported that anti-hypertrophic effect of SMV may due to opposing to the harmful positive inotropic effect of

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β-adrenergic stimulation(33, 34) in ISO-induced cardiac hypertrophy, indicating that the enhancement of inhibitive effects of the SMV-Lipo on cardiac hypertrophy may result from the

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enhanced antagonism effect of the SMV on β-adrenergic stimulation. In addition, the remodeling of extra cellular matrix (ECM), including degradation of the matrix and accumulation of collagen, results in loss of functional cardiomyocytes, declined cardiac

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compliance and compromised diastolic function, which can eventually lead to cardiac

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dysfunction and heart failure(35-37). Myocardial fibrosis is characterized by abnormally high values of myocardial collagen volume fraction, and the percentage of total myocardial tissue occupied by collagen fibers(38, 39). The accumulation of excessive collagen type III fibers within the myocardium results in cardiac fibrosis(40). Our data of the picric sirius red staining revealed that, when given at an equal dose by i.g. administration, it was the SMV-Lipo but not the SMV that significantly decreased the percentage of the fibrosis area. Moreover, at i.g. administration, the SMV-Lipo significantly down-regulated the relative expression of mRNA of collagen-III while there was no difference between the SMV group and the model group. Dramatically, i.p. administration of the SMV-Lipo had a better inhibitive effect on cardiac fibrosis, compared with the double-dosage of i.g. administration, indicating that i.p. was more

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ACCEPTED MANUSCRIPT efficient than i.g. administration. Our results revealed a more significant protective effect of the SMV-Lipo than the SMV on cardiac fibrosis.

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Additionally, inflammation plays a crucial role in adverse cardiac remodeling (41), and macrophages mediate the development of left ventricular hypertrophy and aggravate cardiac remodeling. It has been demonstrated that macrophage depletion significantly down-

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regulates the expression of genes associated with cardiac remodeling and reduces the left ventricular hypertrophy (42). With experimental data showing that statin significantly

anti-inflammatory

effects

(43).

Consistently,

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results

of

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staining,

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direct

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reduces macrophages content within atherosclerotic plaques, SMV has been proved to have

immunohistochemical staining of Mac-2 and relative mRNA expression of F4/80 demonstrate

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that the SMV-Lipo distinctly repressed the infiltration of macrophages in the cardiac interstitial and significantly down-regulated the mRNA expression of F4/80, while macrophage infiltration of the SMV group did not differ from the model group. Moreover, i.p. administration of the SMV-Lipo, even given at a half dosage of i.g. administration, showed

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more significant anti-inflammatory effects than i.g. administration, indicating i.p. was more

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efficient than i.g. administration. All these results prove the predominance of the SMV-Lipo over SMV in anti-inflammation. We next investigated the two typical fetal cardiac genes, ANF and BNP, which are typically silenced in the adult hearts, but can be activated on the condition of pressure overload or myocardial stretch(44). The reactivation of ANF and BNP can be one of the typical characters in cardiac remodeling, as the expression level of ANF and BNP indicates the cardiac function and signifies the prognosis of heart failure patients. Our results illustrate that the SMV-Lipo significantly down-regulated the mRNA expression of ANF and BNP, while there was no statistical difference between the SMV and the model group. This could also be a proof of the more effective amelioration of the cardiac function and the improvement of the prognosis in 15

ACCEPTED MANUSCRIPT the cardiac remodeling by the SMV-Lipo than the SMV, as well as the more efficient results of i.p. administration than i.g. administration.

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Altogether, the SMV-Lipo, when applied at the same dosage and administration regimen, more significantly prevented ISO-induced cardiac remodeling than the SMV. Therefore, by nanoliposome formulation, the absorption of SMV was significantly improved, the great

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therapeutic effects of the SMV was achieved successfully either by i.p. or i.g. and the side

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effects of SMV may be minimized through lowering the dosage of SMV. Based on the multiple pharmacological effects of SMV, the nanoliposome formulation, as an

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efficient SMV delivery system, would not only guarantee the therapeutic effects of SMV but also increase its safety as well as compliance of the patients who have chronic diseases and

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need long-term medical care. And all these benefits make SMV-Lipo a promising practical

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strategy to be applied in clinic to treat multiple diseases in the future.

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prognosis. Circulation 2000;102(4):470-9. 2. Mann DL, Bristow MR. Mechanisms and models in heart failure: the biomechanical model and beyond. Circulation 2005;111(21):2837-49.

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3. Porter KE, Turner NA. Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther 2009;123(2):255-78.

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4. Kitagawa Y, Yamashita D, Ito H, Takaki M. Reversible effects of isoproterenol-induced

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Figure legends Figure 1 Effects of the SMV and the SMV-Lipo on ISO-induced cardiac hypertrophy in vivo. A. HW/BW ratio; B. HW/TI ratio; *** P<0.001 vs. the control group; # P<0.05, ## P<0.01 vs. the

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ISO model group.

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Figure 2 Effects of the SMV and the SMV-Lipo on cardiac function. A. Representative images of echocardiography; B. Diastolic left ventrical posterior wall thickness (LVPW, d); C. Ejection

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fraction (EF%); D. Percent fractional shortening (FS%); E. Left ventrical internal diameter at end-systole (LVID, s) F. Left ventrical internal diameter at end-diastole (LVID, d); *** P<0.001

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vs. the control group; # P<0.05, ## P<0.01 vs. the ISO model group. Figure 3 Relative mRNA expression of ANF (A) and BNP (B); ** P<0.01, *** P<0.001 vs. the

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control group; # P<0.05 vs. the ISO model group. Figure 4 Effects of the SMV and the SMV-Lipo on ISO-induced cardiac fibrosis. A.

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Representative micrographs of the left ventricle (LV) sections stained by picric sirius red. B. Quantitation of picric sirius red staining provided the average interstitial collagen content. C: Relative expression of collagen-III normalized to β-actin. *** P<0.001 vs. the control group; # P<0.05, ### P<0.001 vs. the ISO model group; $$ P<0.01 vs. SMV i.p. administration. Figure 5 Effects of the SMV and the SMV-Lipo on inflammation of the heart. A: Representative micrographs of HE stained sections of the left ventricle (LV). B: Representative micrographs of inmmunohistochemical staining of Mac-2. C: Relative expression of F4/80, normalized to βactin. ** P<0.01 vs. the control group; # P<0.05 vs. the ISO model group. Figure 6 Stable plasma SMV concentrations of the SMV group and the SMV-Lipo group after 22

ACCEPTED MANUSCRIPT i.p. or i.g. administration for 7 days. * P<0.05, ***P<0.001 vs. SMV i.g. (20 mg/kg); ### P<0.001 vs. SMV i.p. (10 mg/kg). Supplemental Figure 1 Effects of the SMV and the SMV-Lipo on the normal mice hearts. A.

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Supplemental Figure 2 Effects of the SMV and the SMV-Lipo on cardiac function of the

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ventricular internal diameter at end-diastole (LVID, d).

Supplemental Figure 3 Effects of the SMV and the SMV-Lipo on cardiac morphology and

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extra cellular matrix of the normal mice. A & B: Representative micrographs of HE (A) and

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picric sirius red (B) stained sections of the left ventricle.

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ACCEPTED MANUSCRIPT Table 1. The groups and drug administration regimens for investigation of the effects of SMV and SMV-Lipo on the ISO-induced mice cardiac remodeling.

Saline

SMV

SMV-Lipo

Control

+

-

+

-

-

ISO

-

+

+

-

-

SMV

-

+

-

+

-

SMV-Lipo

-

+

-

-

+

Control

+

-

+

-

-

ISO

-

+

+

-

-

SMV

-

+

-

+

-

SMV-Lipo

-

+

-

-

+

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ISO

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i.g.

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i.p.

Drug administration regimen

IP T

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Group

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ACCEPTED MANUSCRIPT Table 2. Encapsulation efficiency, particle size, PDI and zeta potential of the SMV-Lipo.

Encapsulation efficiency (%)

Drug loading (g/g, %)

Average particle size (nm)

PDI

SMV-Lipo

95.98 ± 1.35

7.33 ± 0.62

121.4 ± 5.5

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Zeta potential (mV)

−4.5 ± 0.9

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Graphical abstract

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The SMV-Lipo significantly improved plasma concentrations of SMV either by i.p. or i.g. administration, and therefore, more significantly inhibited cardiac hypertrophy, fibrosis and inflammation induced by ISO in mice than the crude SMV.

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