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RGD modified and PEGylated lipid nanoparticles loaded with puerarin: Formulation, characterization and protective effects on acute myocardial ischemia model
Biomedicine & Pharmacotherapy 89 (2017) 297–304
Available online at
ScienceDirect www.sciencedirect.com
Original article
RGD modified and PEGylated lipid nanoparticles loaded with puerarin: Formulation, characterization and protective effects on acute myocardial ischemia model Zhaoqiang Donga,* , Jing Guob , Xiaowei Xinga , Xuguang Zhanga , Yimeng Dua , Qinghua Lua a b
Department of Cardiology, The Second Hospital of Shandong University, Ji’nan, 250033, Shandong, PR China Department of Radiology, The Second Hospital of Shandong University, Ji’nan, 250033, Shandong, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2016 Received in revised form 2 February 2017 Accepted 9 February 2017
Context: Puerarin has been widely used as a therapeutic agent for the treatment of cardiovascular diseases. However, its rapid elimination half-life in plasma and poor water solubility limits its clinical efficacy. Objective: RGD modified and PEGylated solid lipid nanoparticles loaded with puerarin (RGD/PEG-PUESLN) were developed to improve bioavailability of PUE, to prolong retention time in vivo and to enhance its protective effect on acute myocardial ischemia model. Methods: In the present study, RGD-PEG-DSPE was synthesized. RGD/PEG-PUE-SLN were prepared by the solvent evaporation method with some modifications. The physicochemical properties of NPs were characterized, the pharmacokinetics, biodistribution, pharmacodynamic behavior of RGD/PEG-PUE-SLN were evaluated in acute MI rats. Results: The mean diameter, zeta potential, entrapment efficiency and drug loading capacity for RGD/ PEG-PUE-SLN were observed as 110.5 nm, 26.2 mV, 85.7% and 16.5% respectively. PUE from RGD/PEGPUE-SLN exhibited sustained drug release with a burst release during the initial 12 h and a followed sustained release. Pharmacokinetics results indicated that AUC increased from 52.93 (mg/mL h) for free PUE to 176.5 (mg/mL h) for RGD/PEG-PUE-SLN. Similarly, T1/2 increased from 0.73 h for free PUE to 2.62 h for RGD/PEG-PUE-SLN. RGD/PEG-PUE-SLN exhibited higher drug concentration in the heart and plasma compared with other PUE formulations. It can be clearly seen that the infarct size of RGD/PEG-PUE-SLN is the lowest among all the formulation. Conclusion: We conclude that RGD modified and PEGylated SLN are promising candidate delivery vehicles for cardioprotective drugs in treatment of myocardial infarction. © 2017 Published by Elsevier Masson SAS.
Keywords: Myocardial infarction Puerarin Poly(ethylene glycol) Arginine-glycineasparagine Passive targeting Active targeting
1. Introduction Cardiovascular diseases are one of the main causes of death today, with myocardial infarction (MI) and ischemic heart disease contributing a large share of the deaths reported [1]. In patients with ST-segment elevation acute MI, early reperfusion therapy is a standard strategy to limit MI size [2]. However, myocardial ischemia-reperfusion (IR) induces the generation of reactive oxygen species (ROS), calcium overload, and rapid pH correction,
* Corresponding author at: Department of Cardiology, The Second Hospital of Shandong University, No. 247 Beiyuan Street, Ji'nan, 250033, Shandong Province, PR China. E-mail address: [email protected] (Z. Dong). http://dx.doi.org/10.1016/j.biopha.2017.02.029 0753-3322/© 2017 Published by Elsevier Masson SAS.
all of which cause mitochondrial injury, leading to the necrosis and apoptosis of cardiomyocytes [3]. Puerarin (7, 40 -dihydroxyisoflavone-8b-glucopyranoside, PUE) is a major active ingredient derived from the Chinese medical herb Radix Puerariae and widely used as a therapeutic agent for the treatment of cardiovascular diseases. The molecular mechanism underlying these clinical benefits is believed to involve its ability to act as an antioxidant and a scavenger of ROS [4,5]. PUE injection has been approved by the China Food and Drug Administration (CFDA) and used in the treatment of various cardiovascular diseases such as coronary heart disease, MI, arteriosclerosis and arrhythmia [6]. Whereas, disadvantage properties of PUE hinder its clinical application, including poor water solubility, the rapid clearance rate in vivo (short half-life) and low bioavailability [7,8]. The short half-life of PUE in humans suggests frequent intravenous
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administration of high doses may be needed, possibly leading to severe and acute adverse effects including intravenous hemolysis [9,10]. Therefore, it is essential to engineering a drug delivery system to accumulate PUE in ischemic myocardium. As is well known, MI could cause the enhanced permeability and retention (EPR) effect [11,12]. Based on the pathophysiological change, there are two main strategies for targeting parenteral drugs to the ischemic myocardium: passive targeting and active targeting strategies [13–16]. Passive targeting via the EPR effect involves the PEGylated method or nano-sized drug delivery systems such as PEGylated liposomes, PEG-PE micelles, monoPEGylated conjugates, liposomes, PLGA nanoparticles, silica nanoparticles, core-shell hybrid liposomal vesicles, lipid nanoparticles, etc [2,18,19]. Specifically, nanocarriers may be used to protect sensitive molecules from degradation in circulation, thus prolonging their circulation time. Nanocarriers can also be customised with targeting moieties, thus delivering active ingredients to the ischemic myocardium [20–22]. In tissue ischaemia, avb3 integrin, which is highly expressed on endothelial cells during angiogenesis, has been identified as a favorable target for MI treatment [23]. Cyclic arginyl-glycylaspartic acid (RGD) peptide has been explored as a avb3 integrin receptor-specific targeting moiety for the targeted delivery of nanoparticle-loaded therapeutics [24]. Moreover, cell adhesion ligands such as arginine-glycineasparagine (RGD) sequences can be immobilized on nanocarriers to promote cell anchorage [15]. RGD modified nanocarriers can influence the cardiac microenvironment on the molecular level, and enhance angiogenesis [16,25]. Investigators have devoted considerable study to ligands of the integrin for MI therapy. Yu et al. demonstrated that intramyocardial injections of RGD modified alginate could enhance angiogenesis [26]. However, direct myocardial injection is highly invasive, volume-limited and has the potential to cause further injury to the already-weakened myocardium. Therefore, we fabricated a novel RGD modified and PEGylated solid lipid nanoparticles loaded with PUE (RGD/PEG-PUE-SLN) for targeting cardiac cells and treating MI. The SLN were characterized on the basis of morphology, mean diameter, polydispersity index (PDI), zeta potential and entrapment efficiency. In vitro drug release and stability in the fresh plasma were evaluated. In vivo pharmacokinetics biodistribution and pharmacodynamics (infarct size) were investigated in rats with normal or ischemic myocardium after intravenous injection.
2. Materials and methods
2.3. Animals All experimental protocols for animals were performed on Male Sprague-Dawley (SD) rats weighing 220 20 g that were purchased from the Center of Experimental Animals of Shandong Province (China). Animals were housed at 25 2 C with 12 h light/ dark cycles. All animal experiments conformed to the guidelines of the National Act on the Use of Experimental Animals (People’s Republic of China). 2.4. Synthesis of RGD-PEG-DSPE RGD-PEG-DSPE was synthesized by the one step method where the amino group of RGD was conjugated with activated DSPEPEG2000-NHS in the presence of triethylamine (TEA) [27,28]. Briefly, 60 mg of RGD were dissolved in anhydrous N,N-Dimethyl formamide. Under stirring, TEA equivalent to RGD, and 500 mg of DSPE-PEG2000-NHS were added, and the solution was stirred overnight at room temperature. Unreacted RGD was removed by dialysis against deionized water (MV, 3500 Da) for 48 h at room temperature. Finally, RGD-PEG-DSPE was obtained by freezedrying. 2.5. Formulation of SLN The RGD/PEG-PUE-SLN were formulated by the solvent evaporation method with some modifications (Fig. 1) [29,30]. Puerarin (10 mg), 888 ATO (60 mg), injectable soya lecithin (60 mg) and RGD-PEG-DSPE (15 mg) were dissolved and melted in 10 mL ethanol at 55–60 C to form the lipid phase. And the aqueous phase was composed of 0.5% poloxamer 188 (w/v) in double-distilled water and kept at 55–60 C. The lipid phase was injected into the hot aqueous phase (55–60 C) under stirring at 600 rpm until the organic solvent was removed. PEG-PUE-SLN were formulated in the same way as “formulation of RGD/PEG-PUE-SLN” except that RGD-PEG-DSPE was instead of PEG-DSPE. PUE-SLN were formulated in the same way as “formulation of RGD/PEG-PUE-SLN” without RGD-PEG-DSPE. Blank SLN were formulated in the same way as “formulation of PUE-SLN” without PUE. 2.6. Characterization of SLN The mean diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) technology using
2.1. Materials RGD peptide was supplied by the GL Biochem Ltd (Shanghai, China). DSPE-PEG2000-NHS and DSPE-PEG2000 were purchased from Xi’an ruixi Biological Technology Co., Ltd (Xi’an, China). Puerarin was supplied by Reyoung pharmaceutical Co, Ltd (Zibo, China). COMPRITOL1 888 ATO (888 ATO) was kindly provided by Gattefosse’ (Paramus, NJ). Injectable soya lecithin was purchased from Shanghai Taiwei Pharmaceutical Co, Ltd (Shanghai, China).
2.2. Cells Human Cardiac Myocytes (HCM) were obtained from the American type culture collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Fisher Chemicals, Fairlawn, NJ) in a 5% CO2 fully humidified atmosphere.
Fig. 1. A scheme of the structure of RGD/PEG-PUE-SLN.
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Malvern Zetasizer Nano1 Instruments (Malvern, UK). Zeta potential was measured by electrophoretic mobility using the same equipment. All samples were determined in triplicate. PUE concentration was measured at 250 nm by the HPLC method [8]. Samples were chromatographed through a Phenomenex Luna C18 column (4.6 mm 250 mm, 5 mm). Mobile phase consisted of a mixture of 0.1% citric acid in water and methanol (25:75, v/v) with 1.0 mL/min flow rate at 35 C. To determine encapsulation efficacy (EE) and drug loading (DL), desired amount of SLN was centrifuged for 15 min at 3000 rpm at 4 C. Further, the filtrate was removed and injected (20 mL) into HPLC to determine the amount of un-entrapped drug. Equal amount of SLN was dissolved in chloroform: methanol (1:1 v/v), and then centrifuged at 15,000 rpm for 10 min. The supernatant solution was removed and injected (20 mL) into HPLC to determine the amount of total drug. EE% and DL% were calculated using the following formula:
with 3% isofluorane, incubated and underwent endotracheal intubation for mechanical ventilation. The heart was exposed by a left lateral incision through the fourth intercostal space. MI was created by ligation of the left descending coronary artery (LAD). The thorax was closed layer by layer immediately. Ischemia conditions were verified by both ST-segment elevation and changes in the electrocardiography (ECG). There were six groups (n = 6 for each group): sham (thoracotomy without ligation), saline, RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN and free PUE. The rats of PUE loaded SLN or free PUE were treated with the respective drugs at a dose of 50 mg/kg via intravenous injection [36,37]. 2.10. Pharmacokinetics and distribution study After treatment with RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUESLN or free PUE (50 mg/kg), blood samples were obtained at 0, 5, 15,
EEð%Þ ¼
the amount of total drug the amount of unentrapped drug 100 the amount of total drug
DLð%Þ ¼
the amount of total drug the amount of unentrapped drug 100 the amount of lipid þ ðthe amount of total drug the amount of unentrapped drugÞ
2.7. In vitro drug release The in vitro release behavior of PUE from RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN and free PUE was performed using dialysis method in phosphate buffer solutions (PBS) at pH 7.4 under 37 C [31]. 5 mL PUE loaded SLN or free PUE (PUE solution) was enclosed in a dialysis bag and immersed in 100 mL dissolution medium at a stirring rate of 100 rpm. Periodically, samples (0.5 mL) were removed from the release media and analyzed by the HPLC method. After sampling, the release medium was replaced by fresh PBS immediately. 2.8. In vitro cytotoxicity The cytotoxicity of PUE loaded SLN and free PUE were assessed in HCM by a standard MTT assay [32]. In brief, cells were seeded in 48-well plates at 105 cells/well and pre-incubated for 24 h and then incubated with different concentration (1, 5, 10, 50, 100 mM) of RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN, free PUE and 0.9% saline solution for 48 h. Afterwards, cells were washed with PBS for three times and treated with MTT solution (5 mg/mL), then maintained for another 4 h. The MTT medium was removed and formazan crystals dissolved by adding 200 mL DMSO into each well. The relative cell viability is proportional to the absorbance and the untreated cell control was used to approximate to 100% cells viability calculated using equation: Cell viability (%) = Absorbance of Sample/Absorbance of control 100. 2.9. Acute MI model Acute MI model was induced by the proximal left coronary artery ligation method [2,32–34]. Briefly, rats were anesthetized
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30, and 45 min, and at 1, 1.5, 2, 4, 6, 8, 12 and 16 h after i.v. to evaluate pharmacokinetics behaviors. All samples were stored at 20 C for further analysis. For tissue distribution evaluation, rats were received the same recipes as “pharmacokinetics study”. After 3 h, rats were sacrificed to collect samples from the heart, liver, spleen, lung, kidney and brain. All of the tissue specimens were washed, weighed and homogenized, then stored at 20 C for later analysis.
2.11. Plasma or tissue sample treatment Plasma or tissue homogenates (100 mL) were mixed with 20 mL of tectoridin (7.5 mg/mL) as the internal standard and 300 mL of methanol [6,37,38]. The mixture was vortexed for 3 min and centrifuged at 10,000 rpm for 10 min to precipitate the proteins. The supernatant was evaporated in a water bath at 40 C under a stream of nitrogen. The dry sample was redissolved in the mobile phase (100 mL), and analyzed by the HPLC method.
2.12. In vivo efficacy evaluation Rats were randomly divided into six groups as the section of “acute MI model”. Myocardial infarct size was determined by TTC to evaluate in vivo efficacy of PUE loaded SLN or free PUE [2,39,40]. In brief, animals were sacrificed at 36 h after intravenous injection. Then, the hearts were frozen, sliced into 2 mm thick sections parallel to the atrioventricular groove. Slices were incubated with 2% TTC solution for 30 min. Healthy tissue (brick red) and infarct tissue (unstained, pale white) areas were subjected to morphometric analysis after taking pictures. The infarct size was presented as: percentage of infarct size = infarct size/the size of the whole left ventricle 100%.
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SLN ranged between 26 and 33 mV. EE of RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN were 85.7 2.7, 88.9 3.1% and 92.2 2.8% respectively. DL of RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN were 16.5 0.36%, 17.8 0.27% and 18.3 0.19% respectively. 3.3. In vitro drug release The in vitro release behavior was performed using dialysis method in PBS at pH 7.4 under 37 C. As shown in Fig. 3, PUE loaded SLN (RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN) exhibited a burst release during the initial 12 h (56.7%, 60.1% and 80.5% respectively) and a followed sustained release (76.9%, 81.2% and 97.3% respectively in 48 h). 3.4. In vitro cytotoxicity In vitro cytotoxicity of PUE loaded SLN and free PUE at various concentrations was showed in Fig. 4. The cell viabilities of the PUE loaded SLN over the studied concentrations were over 80% and 100% compared with control. RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN groups exhibited no obvious cytotoxicity than the saline control group (p > 0.05).
Fig. 2. 1H NMR spectroscopy of RGD-PEG-DSPE.
2.13. Statistical analysis All data are presented as means standard derivation (SD). Statistical analyses were carried out by two-tailed Student's t-tests or one-way analysis of variance (ANOVA). Pharmacokinetics parameters were calculated using drug and statistics (DAS) version 3.2 software. The criterion for statistical significance was p < 0.05 or p < 0.01. 3. Results 3.1. Synthesis of RGD-PEG-DSPE The structure of RGD-PEG-DSPE was confirmed by 1H NMR spectroscopy (Fig. 2). 1H NMR (DMSO-d6, 300 MHz) d: 2.51 ( NHCO), 3.35 ( CH2N ), 3.48–3.78 (CH2 in PEG), 4.28 ( NH CO ), 4.35–4.49 (NH CH (CH2) CO belongs to RGD). 3.2. Formulation and characterization of SLN In order to improve the pharmacokinetics and pharmacodynamics activity of PUE on myocardial infarction, RGD modified and PEGylated solid lipid nanoparticles loaded with PUE (RGD/PEGPUE-SLN) were successfully fabricated by the solvent evaporation method. RGD/PEG-PUE-SLN were consist of 888 ATO and soya lecithin as the solid core, and PEG-DSPE as the stabilizer and RGD as the target ligand to increase the circulation time and drug accumulation in the infarct area. Table 1 list the characterization data for mean diameter, PDI, zeta potential, EE, and DL of the SLN. The mean diameter was in the range of 70–110 nm with values of the PDI lower than 0.3. The size of PUE-SLN and blank SLN is around 70 nm, while the size of RGD/ PEG-PUE-SLN and PEG-PUE-SLN is around 110 nm. Zeta potential of
3.5. Pharmacokinetics and distribution study Plasma concentrations of PUE vs. time obtained after i.v. administration of different formulations at dose of 50 mg/kg are presented in Fig. 5 and the pharmacokinetics parameters are shown in Table 2. Both the areas under the concentration-time curve (AUC) for the different formulations and T1/2 were significantly different (p < 0.05 or p < 0.01). AUC increased from 52.93 (mg/mL h) for free PUE to 83.17 (mg/mL h) for PUE-SLN, 141.37 (mg/mL h) for PEG-PUE-SLN and 176.5 (mg/mL h) for RGD/PEGPUE-SLN. Similarly, T1/2 increased from 0.73 h for free PUE to 1.35 h for PUE-SLN, 2.37 h for PEG-PUE-SLN and 2.62 h for RGD/PEG-PUESLN. The biodistribution of different PUE formulations was measured at 3 h after i.v. administration in six different organs, including the circulatory system (Fig. 6). RGD/PEG-PUE-SLN exhibited maximum drug concentration in the heart and plasma compared with other PUE formulations. In the acute MI heart tissue, RGD/PEG-PUE-SLN showed a 23.1-fold increase in PUE concentration in comparison to free PUE, 2.2-fold increase in comparison to PUE-SLN, and 1.3-fold increase in comparison to PEG-PUE-SLN. In the plasma, RGD/PEG-PUE-SLN showed an 18.7fold increase in PUE concentration in comparison to free PUE, 3.6fold increase in comparison to PUE-SLN, and 1.2-fold increase in comparison to PEG-PUE-SLN. 3.6. In vivo efficacy evaluation Figs. 7 and 8 show the effect of PUE from different formulations on the myocardial infarct size. It can be clearly seen that the infarct size of RGD/PEG-PUE-SLN is the lowest among all the formulation,
Table 1 Characterization of SLN. Systems
Mean diameter (nm)
PDI
Zeta Potential (mV)
EE (%)
DL (%)
RGD/PEG-PUE-SLN PEG-PUE-SLN PUE-SLN Blank SLN
110.5 3.4 106.3 2.9 72.8 3.1 70.4 1.6
0.23 0.03 0.25 0.04 0.27 0.06 0.21 0.02
26.2 1.8 29.8 2.3 29.3 2.1 33.0 2.6
85.7 2.7 88.9 3.1 92.2 2.8 –
16.5 0.36 17.8 0.27 18.3 0.19 –
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Fig. 3. In vitro release behavior of RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN, and free PUE.
Fig. 4. In vitro cytotoxicity of PUE loaded SLN and free PUE at various concentrations.
Fig. 5. Plasma concentrations of PUE vs. time after i.v. administration of different formulations.
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Table 2 Pharmacokinetic parameters of PUE loaded SLN and free PUE (n = 6). Parameters
RGD/PEG-PUE-SLN
PEG-PUE-SLN
PUE-SLN
Free PUE
Cmax (mg/mL) AUC 0-1 (mg/mL. h) T1/2 (h) MRT 0-1 (h)
115.13 6.22* 176.5 8.63** 2.62 0.15** 2.09 0.13**
112.52 5.16* 141.37 8.63** 2.37 0.13** 1.87 0.12**
108.33 4.03 83.17 5.36* 1.35 0.09* 1.24 0.06*
101.20 3.17 52.93 4.27 0.73 0.04 0.68 0.03
The mark “ The mark “
* **
” represents the p value is lower than 0.05 (p < 0.05). ” represents the p value is lower than 0.01 (p < 0.01).
Fig. 6. The biodistribution of different PUE formulations at 3 h after i.v. administration in six different organs, including the circulatory system.
Fig. 7. The images of RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN, and free PUE on the myocardial infarct size.
Fig. 8. The effect of PUE from different formulations on the myocardial infarct size(* means p < 0.05).
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which is 6.2% (p < 0.05). The infarct size is 18.1%, 28.3%, or 36.0% for PEG-PUE-SLN, PUE-SLN, or free PUE, respectively. 4. Discussion Our present study aimed at exploring a RGD modified and PEGylated solid lipid nanoparticles loaded with PUE for the treatment of myocardial ischemia. It has been reported that targeted nanoparticles provided an attractive approach as they could be injected intravenously, keep long circulation in the body and only bind at the ischemic zone [41,42]. In order to achieve successful targeting goal, there are mainly two strategies: passive targeting by the enhanced permeation and retention effect (EPR) and active targeting via a ligand to the ischemic area [43,44]. Researches have revealed that SLN are gaining importance due to numerous advantages including the capability of poorly soluble drug loading, parenteral administration, sustained release and functionalization over the SLN outlayer [45]. Therefore, our system devised was based on the two aforementioned ways, the first being passive targeting achieved by the smaller particle size of SLN and EPR effect in the ischemic area, and the other being active targeting (RGD) of avb3 receptors in the heart after infarction. Physical characteristics such as the particle size and morphology of the SLN could affect its accumulation in ischemic myocardium, where vascular permeability is enhanced [2,46]. Paulis et al. characterized the distribution pattern of different sized long-circulating lipid-based nanoparticles (micelles, 15 nm; liposomes 100 nm) [47]. Results demonstrated that liposomes displayed slower and more restricted extravasation from the vasculature. In our design, the SLN showed a size in the range of 70–110 nm, in which SLN could accumulate in ischemic myocardium and avoid extravasation from the vasculature. Larger size of RGD/PEG-PUE-SLN and PEG-PUE-SLN (110 nm) than the size of PUE-SLN and blank SLN (70 nm) may due to the PEG modification that enlarged the size of the particles. Zeta potential is a critical evaluation parameter to assess the desired properties of SLN, which could affect the in vivo stability and the platelet function [48,49]. Recently, Fuentes et al. have systemically investigated the relationship between different surface charges of nanoparticles and their platelet aggregation associated with thrombus formation and cardiovascular disease [49]. Results indicated that charged nanoparticles (anionic and cationic) presented higher inhibitions of the platelet aggregation compared to neutral nanoparticles and were beneficial to protect the heart. In our design, the SLN showed a zeta potential between 26 and 33 mV, which could enhance the in vivo stability and inhibit platelet activation. EE and DL are critical parameters to reflect the reasonability of SLN’s formulation and process. The higher EE and of SLN showed that all of SLN had good drug loading capacity. Compared to PUESLN, the DL of PEG-PUE-SLN was lower, and that of RGD/PEG-PUESLN were the lowest; but all DL were above 10 percent. This phenomenon may be due to part of lipid which was substituted by the PEG-DSPE or RGD-PEG-DSPE thus decreasing the loading of hydrophobic drug [50]. Compared with free PUE, the drug-release curves of PUE loaded SLN showed biphasic release (the initial rapid release and followed sustained release) of PUE from all SLN. This early phenomenon may be due to the drug being located on or near the surface of the SLNs. As the diffusion distance between SLN and the release medium increased, the later release profile showed sustained release [51]. Statistically significant difference was observed in PUE release from PEG-PUE-SLN and PUE-SLN (p < 0.05), which may be attributed to the PEG layer furtherly increased the drug release distance. Also the PEG coating here may created a more stable nanoparticle structure that could cause a slower drug release [6].
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RGD/PEG-PUE-SLN had superior physicochemical properties than other SLN formulations and the free PUE. Therefore, further pharmacokinetics and biodistribution in rats were assessed. Pharmacokinetics and biodistribution results showed similar long-circulating characteristics of RGD/PEG-PUE-SLN as that of PEG-PUE-SLN, which proved that SLN elongates the circulation time of RGD in serum and RGD/PEG-PUE-SLN has great potential to accumulate in the myocardial infarct area [6]. The main aim of the PEGylation is to help PUE-SLN to escape from phagocytosis and capture by various organs. Researches demonstrated that PEGylated nanoparticles (100 nm) had a longer circulation in vivo compared with naked nanoparticles [52,53]. The aim of the RGD modification is to deliver more drugs to the infarct zone. RGD/PEGPUE-SLN exhibited higher heart PUE concentration in comparison with PEG-PUE-SLN or free PUE in MI rats (p < 0.05). This phenomenon could be due to passive targeting of PEGylated SLN and active targeting of RGD decoration [13–16]. Infarct size is one of the important indices for evaluating the drug therapeutic effect in the acute MI. In this study, compared with free PUE, RGD modified and PEGylated SLN showed the smallest infarct size and the best therapy. In general, PEGylated lipid nanoparticles are reported to remain in the circulation for up to 48 h, increasing the opportunity for an efficient target-ligand interaction [54–56]. Our pharmacokinetics and pharmacodynamics results are consistence with other PEGylated or RGD modified nanoparticles researches [13–16]. 5. Conclusion In summary, this study demonstrated delivery of RGD/PEGPUE-SLN to acute myocardial infarcts. Based on the beneficial pharmacokinetics and distribution results, we conclude that RGD modified and PEGylated SLN are promising candidate delivery vehicles for cardioprotective drugs in treatment of myocardial infarction. Acknowledgment The work was supported by the Natural Science Foundation of Shandong Province, China (Grant No. ZR2014HL011). References [1] Writing Group Members, D. Mozaffarian, E.J. Benjamin, A.S. Go, D.K. Arnett, M. J. Blaha, M. Cushman, S.R. Das, S. de Ferranti, J.P. Després, H.J. Fullerton, V.J. Howard, M.D. Huffman, C.R. Isasi, M.C. Jiménez, S.E. Judd, B.M. Kissela, J.H. Lichtman, L.D. Lisabeth, S. Liu, R.H. Mackey, D.J. Magid, D.K. McGuire, E.R. Mohler 3rd, C.S. Moy, P. Muntner, M.E. Mussolino, K. Nasir, R.W. Neumar, G. Nichol, L. Palaniappan, D.K. Pandey, M.J. Reeves, C.J. Rodriguez, W. Rosamond, P.D. Sorlie, J. Stein, A. Towfighi, T.N. Turan, S.S. Virani, D. Woo, R.W. Yeh, M.B. Turner, American Heart Association Statistics Committee, Stroke Statistics Subcommittee, Heart Disease and Stroke Statistics- Update: A Report From the American Heart Association, Circulation 133 (January (4)) (2016) e38–360. [2] S. Zhang, J. Wang, J. Pan, Baicalin-loaded PEGylated lipid nanoparticles: characterization, pharmacokinetics, and protective effects on acute myocardial ischemia in rats, Drug Deliv. 23 (November (9)) (2016) 3696–3703. [3] Y. Nakano, T. Matoba, M. Tokutome, D. Funamoto, S. Katsuki, G. Ikeda, K. Nagaoka, A. Ishikita, K. Nakano, J. Koga, K. Sunagawa, K. Egashira, Nanoparticle-mediated delivery of irbesartan induces cardioprotection from myocardial ischemia-reperfusion injury by antagonizing monocyte-mediated inflammation, Sci. Rep. 11 (July (6)) (2016) 29601. [4] R.M. Han, Y.X. Tian, E.M. Becker, M.L. Andersen, J.P. Zhang, L.H. Skibsted, Puerarin and conjugate bases as radical scavengers and antioxidants: molecular mechanism and synergism with beta-carotene, J. Agric. Food Chem. 55 (March (6)) (2007) 2384–2391 (21). [5] Y. Gao, X. Wang, C. He, An isoflavonoid-enriched extract from Pueraria lobata (kudzu) root protects human umbilical vein endothelial cells against oxidative stress induced apoptosis, J. Ethnopharmacol. 4 (December (193)) (2016) 524– 530. [6] X. Liu, Y. Ding, B. Zhao, Y. Liu, S. Luo, J. Wu, J. Li, D. Xiang, In vitro and in vivo evaluation of puerarin-loaded PEGylated mesoporous silica nanoparticles, Drug Dev. Ind. Pharm. 42 (December (12)) (2016) 2031–2037.
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Original article
RGD modified and PEGylated lipid nanoparticles loaded with puerarin: Formulation, characterization and protective effects on acute myocardial ischemia model Zhaoqiang Donga,* , Jing Guob , Xiaowei Xinga , Xuguang Zhanga , Yimeng Dua , Qinghua Lua a b
Department of Cardiology, The Second Hospital of Shandong University, Ji’nan, 250033, Shandong, PR China Department of Radiology, The Second Hospital of Shandong University, Ji’nan, 250033, Shandong, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2016 Received in revised form 2 February 2017 Accepted 9 February 2017
Context: Puerarin has been widely used as a therapeutic agent for the treatment of cardiovascular diseases. However, its rapid elimination half-life in plasma and poor water solubility limits its clinical efficacy. Objective: RGD modified and PEGylated solid lipid nanoparticles loaded with puerarin (RGD/PEG-PUESLN) were developed to improve bioavailability of PUE, to prolong retention time in vivo and to enhance its protective effect on acute myocardial ischemia model. Methods: In the present study, RGD-PEG-DSPE was synthesized. RGD/PEG-PUE-SLN were prepared by the solvent evaporation method with some modifications. The physicochemical properties of NPs were characterized, the pharmacokinetics, biodistribution, pharmacodynamic behavior of RGD/PEG-PUE-SLN were evaluated in acute MI rats. Results: The mean diameter, zeta potential, entrapment efficiency and drug loading capacity for RGD/ PEG-PUE-SLN were observed as 110.5 nm, 26.2 mV, 85.7% and 16.5% respectively. PUE from RGD/PEGPUE-SLN exhibited sustained drug release with a burst release during the initial 12 h and a followed sustained release. Pharmacokinetics results indicated that AUC increased from 52.93 (mg/mL h) for free PUE to 176.5 (mg/mL h) for RGD/PEG-PUE-SLN. Similarly, T1/2 increased from 0.73 h for free PUE to 2.62 h for RGD/PEG-PUE-SLN. RGD/PEG-PUE-SLN exhibited higher drug concentration in the heart and plasma compared with other PUE formulations. It can be clearly seen that the infarct size of RGD/PEG-PUE-SLN is the lowest among all the formulation. Conclusion: We conclude that RGD modified and PEGylated SLN are promising candidate delivery vehicles for cardioprotective drugs in treatment of myocardial infarction. © 2017 Published by Elsevier Masson SAS.
Keywords: Myocardial infarction Puerarin Poly(ethylene glycol) Arginine-glycineasparagine Passive targeting Active targeting
1. Introduction Cardiovascular diseases are one of the main causes of death today, with myocardial infarction (MI) and ischemic heart disease contributing a large share of the deaths reported [1]. In patients with ST-segment elevation acute MI, early reperfusion therapy is a standard strategy to limit MI size [2]. However, myocardial ischemia-reperfusion (IR) induces the generation of reactive oxygen species (ROS), calcium overload, and rapid pH correction,
* Corresponding author at: Department of Cardiology, The Second Hospital of Shandong University, No. 247 Beiyuan Street, Ji'nan, 250033, Shandong Province, PR China. E-mail address: [email protected] (Z. Dong). http://dx.doi.org/10.1016/j.biopha.2017.02.029 0753-3322/© 2017 Published by Elsevier Masson SAS.
all of which cause mitochondrial injury, leading to the necrosis and apoptosis of cardiomyocytes [3]. Puerarin (7, 40 -dihydroxyisoflavone-8b-glucopyranoside, PUE) is a major active ingredient derived from the Chinese medical herb Radix Puerariae and widely used as a therapeutic agent for the treatment of cardiovascular diseases. The molecular mechanism underlying these clinical benefits is believed to involve its ability to act as an antioxidant and a scavenger of ROS [4,5]. PUE injection has been approved by the China Food and Drug Administration (CFDA) and used in the treatment of various cardiovascular diseases such as coronary heart disease, MI, arteriosclerosis and arrhythmia [6]. Whereas, disadvantage properties of PUE hinder its clinical application, including poor water solubility, the rapid clearance rate in vivo (short half-life) and low bioavailability [7,8]. The short half-life of PUE in humans suggests frequent intravenous
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administration of high doses may be needed, possibly leading to severe and acute adverse effects including intravenous hemolysis [9,10]. Therefore, it is essential to engineering a drug delivery system to accumulate PUE in ischemic myocardium. As is well known, MI could cause the enhanced permeability and retention (EPR) effect [11,12]. Based on the pathophysiological change, there are two main strategies for targeting parenteral drugs to the ischemic myocardium: passive targeting and active targeting strategies [13–16]. Passive targeting via the EPR effect involves the PEGylated method or nano-sized drug delivery systems such as PEGylated liposomes, PEG-PE micelles, monoPEGylated conjugates, liposomes, PLGA nanoparticles, silica nanoparticles, core-shell hybrid liposomal vesicles, lipid nanoparticles, etc [2,18,19]. Specifically, nanocarriers may be used to protect sensitive molecules from degradation in circulation, thus prolonging their circulation time. Nanocarriers can also be customised with targeting moieties, thus delivering active ingredients to the ischemic myocardium [20–22]. In tissue ischaemia, avb3 integrin, which is highly expressed on endothelial cells during angiogenesis, has been identified as a favorable target for MI treatment [23]. Cyclic arginyl-glycylaspartic acid (RGD) peptide has been explored as a avb3 integrin receptor-specific targeting moiety for the targeted delivery of nanoparticle-loaded therapeutics [24]. Moreover, cell adhesion ligands such as arginine-glycineasparagine (RGD) sequences can be immobilized on nanocarriers to promote cell anchorage [15]. RGD modified nanocarriers can influence the cardiac microenvironment on the molecular level, and enhance angiogenesis [16,25]. Investigators have devoted considerable study to ligands of the integrin for MI therapy. Yu et al. demonstrated that intramyocardial injections of RGD modified alginate could enhance angiogenesis [26]. However, direct myocardial injection is highly invasive, volume-limited and has the potential to cause further injury to the already-weakened myocardium. Therefore, we fabricated a novel RGD modified and PEGylated solid lipid nanoparticles loaded with PUE (RGD/PEG-PUE-SLN) for targeting cardiac cells and treating MI. The SLN were characterized on the basis of morphology, mean diameter, polydispersity index (PDI), zeta potential and entrapment efficiency. In vitro drug release and stability in the fresh plasma were evaluated. In vivo pharmacokinetics biodistribution and pharmacodynamics (infarct size) were investigated in rats with normal or ischemic myocardium after intravenous injection.
2. Materials and methods
2.3. Animals All experimental protocols for animals were performed on Male Sprague-Dawley (SD) rats weighing 220 20 g that were purchased from the Center of Experimental Animals of Shandong Province (China). Animals were housed at 25 2 C with 12 h light/ dark cycles. All animal experiments conformed to the guidelines of the National Act on the Use of Experimental Animals (People’s Republic of China). 2.4. Synthesis of RGD-PEG-DSPE RGD-PEG-DSPE was synthesized by the one step method where the amino group of RGD was conjugated with activated DSPEPEG2000-NHS in the presence of triethylamine (TEA) [27,28]. Briefly, 60 mg of RGD were dissolved in anhydrous N,N-Dimethyl formamide. Under stirring, TEA equivalent to RGD, and 500 mg of DSPE-PEG2000-NHS were added, and the solution was stirred overnight at room temperature. Unreacted RGD was removed by dialysis against deionized water (MV, 3500 Da) for 48 h at room temperature. Finally, RGD-PEG-DSPE was obtained by freezedrying. 2.5. Formulation of SLN The RGD/PEG-PUE-SLN were formulated by the solvent evaporation method with some modifications (Fig. 1) [29,30]. Puerarin (10 mg), 888 ATO (60 mg), injectable soya lecithin (60 mg) and RGD-PEG-DSPE (15 mg) were dissolved and melted in 10 mL ethanol at 55–60 C to form the lipid phase. And the aqueous phase was composed of 0.5% poloxamer 188 (w/v) in double-distilled water and kept at 55–60 C. The lipid phase was injected into the hot aqueous phase (55–60 C) under stirring at 600 rpm until the organic solvent was removed. PEG-PUE-SLN were formulated in the same way as “formulation of RGD/PEG-PUE-SLN” except that RGD-PEG-DSPE was instead of PEG-DSPE. PUE-SLN were formulated in the same way as “formulation of RGD/PEG-PUE-SLN” without RGD-PEG-DSPE. Blank SLN were formulated in the same way as “formulation of PUE-SLN” without PUE. 2.6. Characterization of SLN The mean diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) technology using
2.1. Materials RGD peptide was supplied by the GL Biochem Ltd (Shanghai, China). DSPE-PEG2000-NHS and DSPE-PEG2000 were purchased from Xi’an ruixi Biological Technology Co., Ltd (Xi’an, China). Puerarin was supplied by Reyoung pharmaceutical Co, Ltd (Zibo, China). COMPRITOL1 888 ATO (888 ATO) was kindly provided by Gattefosse’ (Paramus, NJ). Injectable soya lecithin was purchased from Shanghai Taiwei Pharmaceutical Co, Ltd (Shanghai, China).
2.2. Cells Human Cardiac Myocytes (HCM) were obtained from the American type culture collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Fisher Chemicals, Fairlawn, NJ) in a 5% CO2 fully humidified atmosphere.
Fig. 1. A scheme of the structure of RGD/PEG-PUE-SLN.
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Malvern Zetasizer Nano1 Instruments (Malvern, UK). Zeta potential was measured by electrophoretic mobility using the same equipment. All samples were determined in triplicate. PUE concentration was measured at 250 nm by the HPLC method [8]. Samples were chromatographed through a Phenomenex Luna C18 column (4.6 mm 250 mm, 5 mm). Mobile phase consisted of a mixture of 0.1% citric acid in water and methanol (25:75, v/v) with 1.0 mL/min flow rate at 35 C. To determine encapsulation efficacy (EE) and drug loading (DL), desired amount of SLN was centrifuged for 15 min at 3000 rpm at 4 C. Further, the filtrate was removed and injected (20 mL) into HPLC to determine the amount of un-entrapped drug. Equal amount of SLN was dissolved in chloroform: methanol (1:1 v/v), and then centrifuged at 15,000 rpm for 10 min. The supernatant solution was removed and injected (20 mL) into HPLC to determine the amount of total drug. EE% and DL% were calculated using the following formula:
with 3% isofluorane, incubated and underwent endotracheal intubation for mechanical ventilation. The heart was exposed by a left lateral incision through the fourth intercostal space. MI was created by ligation of the left descending coronary artery (LAD). The thorax was closed layer by layer immediately. Ischemia conditions were verified by both ST-segment elevation and changes in the electrocardiography (ECG). There were six groups (n = 6 for each group): sham (thoracotomy without ligation), saline, RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN and free PUE. The rats of PUE loaded SLN or free PUE were treated with the respective drugs at a dose of 50 mg/kg via intravenous injection [36,37]. 2.10. Pharmacokinetics and distribution study After treatment with RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUESLN or free PUE (50 mg/kg), blood samples were obtained at 0, 5, 15,
EEð%Þ ¼
the amount of total drug the amount of unentrapped drug 100 the amount of total drug
DLð%Þ ¼
the amount of total drug the amount of unentrapped drug 100 the amount of lipid þ ðthe amount of total drug the amount of unentrapped drugÞ
2.7. In vitro drug release The in vitro release behavior of PUE from RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN and free PUE was performed using dialysis method in phosphate buffer solutions (PBS) at pH 7.4 under 37 C [31]. 5 mL PUE loaded SLN or free PUE (PUE solution) was enclosed in a dialysis bag and immersed in 100 mL dissolution medium at a stirring rate of 100 rpm. Periodically, samples (0.5 mL) were removed from the release media and analyzed by the HPLC method. After sampling, the release medium was replaced by fresh PBS immediately. 2.8. In vitro cytotoxicity The cytotoxicity of PUE loaded SLN and free PUE were assessed in HCM by a standard MTT assay [32]. In brief, cells were seeded in 48-well plates at 105 cells/well and pre-incubated for 24 h and then incubated with different concentration (1, 5, 10, 50, 100 mM) of RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN, free PUE and 0.9% saline solution for 48 h. Afterwards, cells were washed with PBS for three times and treated with MTT solution (5 mg/mL), then maintained for another 4 h. The MTT medium was removed and formazan crystals dissolved by adding 200 mL DMSO into each well. The relative cell viability is proportional to the absorbance and the untreated cell control was used to approximate to 100% cells viability calculated using equation: Cell viability (%) = Absorbance of Sample/Absorbance of control 100. 2.9. Acute MI model Acute MI model was induced by the proximal left coronary artery ligation method [2,32–34]. Briefly, rats were anesthetized
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30, and 45 min, and at 1, 1.5, 2, 4, 6, 8, 12 and 16 h after i.v. to evaluate pharmacokinetics behaviors. All samples were stored at 20 C for further analysis. For tissue distribution evaluation, rats were received the same recipes as “pharmacokinetics study”. After 3 h, rats were sacrificed to collect samples from the heart, liver, spleen, lung, kidney and brain. All of the tissue specimens were washed, weighed and homogenized, then stored at 20 C for later analysis.
2.11. Plasma or tissue sample treatment Plasma or tissue homogenates (100 mL) were mixed with 20 mL of tectoridin (7.5 mg/mL) as the internal standard and 300 mL of methanol [6,37,38]. The mixture was vortexed for 3 min and centrifuged at 10,000 rpm for 10 min to precipitate the proteins. The supernatant was evaporated in a water bath at 40 C under a stream of nitrogen. The dry sample was redissolved in the mobile phase (100 mL), and analyzed by the HPLC method.
2.12. In vivo efficacy evaluation Rats were randomly divided into six groups as the section of “acute MI model”. Myocardial infarct size was determined by TTC to evaluate in vivo efficacy of PUE loaded SLN or free PUE [2,39,40]. In brief, animals were sacrificed at 36 h after intravenous injection. Then, the hearts were frozen, sliced into 2 mm thick sections parallel to the atrioventricular groove. Slices were incubated with 2% TTC solution for 30 min. Healthy tissue (brick red) and infarct tissue (unstained, pale white) areas were subjected to morphometric analysis after taking pictures. The infarct size was presented as: percentage of infarct size = infarct size/the size of the whole left ventricle 100%.
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SLN ranged between 26 and 33 mV. EE of RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN were 85.7 2.7, 88.9 3.1% and 92.2 2.8% respectively. DL of RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN were 16.5 0.36%, 17.8 0.27% and 18.3 0.19% respectively. 3.3. In vitro drug release The in vitro release behavior was performed using dialysis method in PBS at pH 7.4 under 37 C. As shown in Fig. 3, PUE loaded SLN (RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN) exhibited a burst release during the initial 12 h (56.7%, 60.1% and 80.5% respectively) and a followed sustained release (76.9%, 81.2% and 97.3% respectively in 48 h). 3.4. In vitro cytotoxicity In vitro cytotoxicity of PUE loaded SLN and free PUE at various concentrations was showed in Fig. 4. The cell viabilities of the PUE loaded SLN over the studied concentrations were over 80% and 100% compared with control. RGD/PEG-PUE-SLN, PEG-PUE-SLN, and PUE-SLN groups exhibited no obvious cytotoxicity than the saline control group (p > 0.05).
Fig. 2. 1H NMR spectroscopy of RGD-PEG-DSPE.
2.13. Statistical analysis All data are presented as means standard derivation (SD). Statistical analyses were carried out by two-tailed Student's t-tests or one-way analysis of variance (ANOVA). Pharmacokinetics parameters were calculated using drug and statistics (DAS) version 3.2 software. The criterion for statistical significance was p < 0.05 or p < 0.01. 3. Results 3.1. Synthesis of RGD-PEG-DSPE The structure of RGD-PEG-DSPE was confirmed by 1H NMR spectroscopy (Fig. 2). 1H NMR (DMSO-d6, 300 MHz) d: 2.51 ( NHCO), 3.35 ( CH2N ), 3.48–3.78 (CH2 in PEG), 4.28 ( NH CO ), 4.35–4.49 (NH CH (CH2) CO belongs to RGD). 3.2. Formulation and characterization of SLN In order to improve the pharmacokinetics and pharmacodynamics activity of PUE on myocardial infarction, RGD modified and PEGylated solid lipid nanoparticles loaded with PUE (RGD/PEGPUE-SLN) were successfully fabricated by the solvent evaporation method. RGD/PEG-PUE-SLN were consist of 888 ATO and soya lecithin as the solid core, and PEG-DSPE as the stabilizer and RGD as the target ligand to increase the circulation time and drug accumulation in the infarct area. Table 1 list the characterization data for mean diameter, PDI, zeta potential, EE, and DL of the SLN. The mean diameter was in the range of 70–110 nm with values of the PDI lower than 0.3. The size of PUE-SLN and blank SLN is around 70 nm, while the size of RGD/ PEG-PUE-SLN and PEG-PUE-SLN is around 110 nm. Zeta potential of
3.5. Pharmacokinetics and distribution study Plasma concentrations of PUE vs. time obtained after i.v. administration of different formulations at dose of 50 mg/kg are presented in Fig. 5 and the pharmacokinetics parameters are shown in Table 2. Both the areas under the concentration-time curve (AUC) for the different formulations and T1/2 were significantly different (p < 0.05 or p < 0.01). AUC increased from 52.93 (mg/mL h) for free PUE to 83.17 (mg/mL h) for PUE-SLN, 141.37 (mg/mL h) for PEG-PUE-SLN and 176.5 (mg/mL h) for RGD/PEGPUE-SLN. Similarly, T1/2 increased from 0.73 h for free PUE to 1.35 h for PUE-SLN, 2.37 h for PEG-PUE-SLN and 2.62 h for RGD/PEG-PUESLN. The biodistribution of different PUE formulations was measured at 3 h after i.v. administration in six different organs, including the circulatory system (Fig. 6). RGD/PEG-PUE-SLN exhibited maximum drug concentration in the heart and plasma compared with other PUE formulations. In the acute MI heart tissue, RGD/PEG-PUE-SLN showed a 23.1-fold increase in PUE concentration in comparison to free PUE, 2.2-fold increase in comparison to PUE-SLN, and 1.3-fold increase in comparison to PEG-PUE-SLN. In the plasma, RGD/PEG-PUE-SLN showed an 18.7fold increase in PUE concentration in comparison to free PUE, 3.6fold increase in comparison to PUE-SLN, and 1.2-fold increase in comparison to PEG-PUE-SLN. 3.6. In vivo efficacy evaluation Figs. 7 and 8 show the effect of PUE from different formulations on the myocardial infarct size. It can be clearly seen that the infarct size of RGD/PEG-PUE-SLN is the lowest among all the formulation,
Table 1 Characterization of SLN. Systems
Mean diameter (nm)
PDI
Zeta Potential (mV)
EE (%)
DL (%)
RGD/PEG-PUE-SLN PEG-PUE-SLN PUE-SLN Blank SLN
110.5 3.4 106.3 2.9 72.8 3.1 70.4 1.6
0.23 0.03 0.25 0.04 0.27 0.06 0.21 0.02
26.2 1.8 29.8 2.3 29.3 2.1 33.0 2.6
85.7 2.7 88.9 3.1 92.2 2.8 –
16.5 0.36 17.8 0.27 18.3 0.19 –
Z. Dong et al. / Biomedicine & Pharmacotherapy 89 (2017) 297–304
Fig. 3. In vitro release behavior of RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN, and free PUE.
Fig. 4. In vitro cytotoxicity of PUE loaded SLN and free PUE at various concentrations.
Fig. 5. Plasma concentrations of PUE vs. time after i.v. administration of different formulations.
301
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Table 2 Pharmacokinetic parameters of PUE loaded SLN and free PUE (n = 6). Parameters
RGD/PEG-PUE-SLN
PEG-PUE-SLN
PUE-SLN
Free PUE
Cmax (mg/mL) AUC 0-1 (mg/mL. h) T1/2 (h) MRT 0-1 (h)
115.13 6.22* 176.5 8.63** 2.62 0.15** 2.09 0.13**
112.52 5.16* 141.37 8.63** 2.37 0.13** 1.87 0.12**
108.33 4.03 83.17 5.36* 1.35 0.09* 1.24 0.06*
101.20 3.17 52.93 4.27 0.73 0.04 0.68 0.03
The mark “ The mark “
* **
” represents the p value is lower than 0.05 (p < 0.05). ” represents the p value is lower than 0.01 (p < 0.01).
Fig. 6. The biodistribution of different PUE formulations at 3 h after i.v. administration in six different organs, including the circulatory system.
Fig. 7. The images of RGD/PEG-PUE-SLN, PEG-PUE-SLN, PUE-SLN, and free PUE on the myocardial infarct size.
Fig. 8. The effect of PUE from different formulations on the myocardial infarct size(* means p < 0.05).
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which is 6.2% (p < 0.05). The infarct size is 18.1%, 28.3%, or 36.0% for PEG-PUE-SLN, PUE-SLN, or free PUE, respectively. 4. Discussion Our present study aimed at exploring a RGD modified and PEGylated solid lipid nanoparticles loaded with PUE for the treatment of myocardial ischemia. It has been reported that targeted nanoparticles provided an attractive approach as they could be injected intravenously, keep long circulation in the body and only bind at the ischemic zone [41,42]. In order to achieve successful targeting goal, there are mainly two strategies: passive targeting by the enhanced permeation and retention effect (EPR) and active targeting via a ligand to the ischemic area [43,44]. Researches have revealed that SLN are gaining importance due to numerous advantages including the capability of poorly soluble drug loading, parenteral administration, sustained release and functionalization over the SLN outlayer [45]. Therefore, our system devised was based on the two aforementioned ways, the first being passive targeting achieved by the smaller particle size of SLN and EPR effect in the ischemic area, and the other being active targeting (RGD) of avb3 receptors in the heart after infarction. Physical characteristics such as the particle size and morphology of the SLN could affect its accumulation in ischemic myocardium, where vascular permeability is enhanced [2,46]. Paulis et al. characterized the distribution pattern of different sized long-circulating lipid-based nanoparticles (micelles, 15 nm; liposomes 100 nm) [47]. Results demonstrated that liposomes displayed slower and more restricted extravasation from the vasculature. In our design, the SLN showed a size in the range of 70–110 nm, in which SLN could accumulate in ischemic myocardium and avoid extravasation from the vasculature. Larger size of RGD/PEG-PUE-SLN and PEG-PUE-SLN (110 nm) than the size of PUE-SLN and blank SLN (70 nm) may due to the PEG modification that enlarged the size of the particles. Zeta potential is a critical evaluation parameter to assess the desired properties of SLN, which could affect the in vivo stability and the platelet function [48,49]. Recently, Fuentes et al. have systemically investigated the relationship between different surface charges of nanoparticles and their platelet aggregation associated with thrombus formation and cardiovascular disease [49]. Results indicated that charged nanoparticles (anionic and cationic) presented higher inhibitions of the platelet aggregation compared to neutral nanoparticles and were beneficial to protect the heart. In our design, the SLN showed a zeta potential between 26 and 33 mV, which could enhance the in vivo stability and inhibit platelet activation. EE and DL are critical parameters to reflect the reasonability of SLN’s formulation and process. The higher EE and of SLN showed that all of SLN had good drug loading capacity. Compared to PUESLN, the DL of PEG-PUE-SLN was lower, and that of RGD/PEG-PUESLN were the lowest; but all DL were above 10 percent. This phenomenon may be due to part of lipid which was substituted by the PEG-DSPE or RGD-PEG-DSPE thus decreasing the loading of hydrophobic drug [50]. Compared with free PUE, the drug-release curves of PUE loaded SLN showed biphasic release (the initial rapid release and followed sustained release) of PUE from all SLN. This early phenomenon may be due to the drug being located on or near the surface of the SLNs. As the diffusion distance between SLN and the release medium increased, the later release profile showed sustained release [51]. Statistically significant difference was observed in PUE release from PEG-PUE-SLN and PUE-SLN (p < 0.05), which may be attributed to the PEG layer furtherly increased the drug release distance. Also the PEG coating here may created a more stable nanoparticle structure that could cause a slower drug release [6].
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RGD/PEG-PUE-SLN had superior physicochemical properties than other SLN formulations and the free PUE. Therefore, further pharmacokinetics and biodistribution in rats were assessed. Pharmacokinetics and biodistribution results showed similar long-circulating characteristics of RGD/PEG-PUE-SLN as that of PEG-PUE-SLN, which proved that SLN elongates the circulation time of RGD in serum and RGD/PEG-PUE-SLN has great potential to accumulate in the myocardial infarct area [6]. The main aim of the PEGylation is to help PUE-SLN to escape from phagocytosis and capture by various organs. Researches demonstrated that PEGylated nanoparticles (100 nm) had a longer circulation in vivo compared with naked nanoparticles [52,53]. The aim of the RGD modification is to deliver more drugs to the infarct zone. RGD/PEGPUE-SLN exhibited higher heart PUE concentration in comparison with PEG-PUE-SLN or free PUE in MI rats (p < 0.05). This phenomenon could be due to passive targeting of PEGylated SLN and active targeting of RGD decoration [13–16]. Infarct size is one of the important indices for evaluating the drug therapeutic effect in the acute MI. In this study, compared with free PUE, RGD modified and PEGylated SLN showed the smallest infarct size and the best therapy. In general, PEGylated lipid nanoparticles are reported to remain in the circulation for up to 48 h, increasing the opportunity for an efficient target-ligand interaction [54–56]. Our pharmacokinetics and pharmacodynamics results are consistence with other PEGylated or RGD modified nanoparticles researches [13–16]. 5. Conclusion In summary, this study demonstrated delivery of RGD/PEGPUE-SLN to acute myocardial infarcts. Based on the beneficial pharmacokinetics and distribution results, we conclude that RGD modified and PEGylated SLN are promising candidate delivery vehicles for cardioprotective drugs in treatment of myocardial infarction. Acknowledgment The work was supported by the Natural Science Foundation of Shandong Province, China (Grant No. ZR2014HL011). References [1] Writing Group Members, D. Mozaffarian, E.J. Benjamin, A.S. Go, D.K. Arnett, M. J. Blaha, M. Cushman, S.R. Das, S. de Ferranti, J.P. Després, H.J. Fullerton, V.J. Howard, M.D. Huffman, C.R. Isasi, M.C. Jiménez, S.E. Judd, B.M. Kissela, J.H. Lichtman, L.D. Lisabeth, S. Liu, R.H. Mackey, D.J. Magid, D.K. McGuire, E.R. Mohler 3rd, C.S. Moy, P. Muntner, M.E. Mussolino, K. Nasir, R.W. Neumar, G. Nichol, L. Palaniappan, D.K. Pandey, M.J. Reeves, C.J. Rodriguez, W. Rosamond, P.D. Sorlie, J. Stein, A. Towfighi, T.N. Turan, S.S. Virani, D. Woo, R.W. Yeh, M.B. Turner, American Heart Association Statistics Committee, Stroke Statistics Subcommittee, Heart Disease and Stroke Statistics- Update: A Report From the American Heart Association, Circulation 133 (January (4)) (2016) e38–360. [2] S. Zhang, J. Wang, J. Pan, Baicalin-loaded PEGylated lipid nanoparticles: characterization, pharmacokinetics, and protective effects on acute myocardial ischemia in rats, Drug Deliv. 23 (November (9)) (2016) 3696–3703. [3] Y. Nakano, T. Matoba, M. Tokutome, D. Funamoto, S. Katsuki, G. Ikeda, K. Nagaoka, A. Ishikita, K. Nakano, J. Koga, K. Sunagawa, K. Egashira, Nanoparticle-mediated delivery of irbesartan induces cardioprotection from myocardial ischemia-reperfusion injury by antagonizing monocyte-mediated inflammation, Sci. Rep. 11 (July (6)) (2016) 29601. [4] R.M. Han, Y.X. Tian, E.M. Becker, M.L. Andersen, J.P. Zhang, L.H. Skibsted, Puerarin and conjugate bases as radical scavengers and antioxidants: molecular mechanism and synergism with beta-carotene, J. Agric. Food Chem. 55 (March (6)) (2007) 2384–2391 (21). [5] Y. Gao, X. Wang, C. He, An isoflavonoid-enriched extract from Pueraria lobata (kudzu) root protects human umbilical vein endothelial cells against oxidative stress induced apoptosis, J. Ethnopharmacol. 4 (December (193)) (2016) 524– 530. [6] X. Liu, Y. Ding, B. Zhao, Y. Liu, S. Luo, J. Wu, J. Li, D. Xiang, In vitro and in vivo evaluation of puerarin-loaded PEGylated mesoporous silica nanoparticles, Drug Dev. Ind. Pharm. 42 (December (12)) (2016) 2031–2037.
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