[Pyr1]-Apelin-13 delivery via nano-liposomal encapsulation attenuates pressure overload-induced cardiac dysfunction

Abstract

Nanoparticle-mediated sustained delivery of therapeutics is one of the highly effective and increasingly utilized applications of nanomedicine. Here, we report the development and application of a drug delivery system consisting of polyethylene glycol (PEG)-conjugated liposomal nanoparticles as an efficient in vivo delivery approach for [Pyr1]-apelin-13 polypeptide. Apelin is an adipokine that regulates a variety of biological functions including cardiac hypertrophy and hypertrophy-induced heart failure. The clinical use of apelin has been greatly impaired by its remarkably short half-life in circulation. Here, we investigate whether [Pyr1]-apelin-13 encapsulation in liposome nanocarriers, conjugated with PEG polymer on their surface, can prolong apelin stability in the blood stream and potentiate apelin beneficial effects in cardiac function. Atomic force microscopy and dynamic light scattering were used to assess the structure and size distribution of drug-laden nanoparticles. [Pyr1]-apelin-13 encapsulation in PEGylated liposomal nanocarriers resulted in sustained and extended drug release both in vitro and in vivo. Moreover, intraperitoneal injection of [Pyr1]-apelin-13 nanocarriers in a mouse model of pressure-overload induced heart failure demonstrated a sustainable long-term effect of [Pyr1]-apelin-13 in preventing cardiac dysfunction. We concluded that this engineered nanocarrier system can serve as a delivery platform for treating heart injuries through sustained bioavailability of cardioprotective therapeutics.

1. Introduction

Cardiac hypertrophy is an adaptive response of the heart cells to elevated levels of biomechanical stress imposed by a variety of extrinsic and intrinsic stimuli including pressure or volume overload, familial/genetic cardiomyopathies, or loss of contractile mass from preceding infarction [1–3]. If sustained, hypertrophy often becomes pathological, accompanied by significant risk of arrhythmia, progression to heart failure, and sudden death [1,4,5]. At the molecular level, pathological hypertrophy is associated with re-induction of the so-called fetal gene program in which the fetal isoforms of genes responsible for regulating cardiac contractility and calcium handling (e.g. β-MHC) are upregulated [1,2,6,7]. At the cellular level, the main characteristics of ventricular hypertrophic growth are enhanced protein synthesis and an increase in size of cardiomyocytes [1,2]. As pathologic hypertrophy progresses, these changes in molecular and cellular phenotypes are accompanied by an increase in apoptosis, fibrosis, chamber dilation, and decreased systolic function [1].

The murine model of transverse aortic constriction (TAC) is one of the most common experimental models used to study pressure overload-induced ventricular hypertrophy and elucidate the key signaling processes involved in the cardiac hypertrophic response and its progress to heart failure [8–11]. TAC initially results in compensated hypertrophy, frequently associated with a transient improvement in myocardial contractility; however, chronic hemodynamic overload leads to maladaptive hypertrophy accompanied by ventricular dilatation and heart failure [8,10,12,13]. The TAC model has been utilized as a platform for examining the utility of pharmacological or molecular interventions that may limit hypertrophy or attenuate the hypertrophy-induced cardiac dysfunction [8,10].

Recent studies have shown that exogenous apelin plays a critical role in ameliorating cardiac dysfunction and/or remodeling in various animal models of cardiac disease such as myocardial infarction, ischemia reperfusion, and hypertrophy (e.g. Refs. [14–16]). Apelin is an endogenous peptide ligand for the G-protein-coupled AplnR (aka APJ), known to be involved in a broad range of physiological functions, including maintaining body fluid homeostasis, blood pressure, obesity, and heart development and function [17–20]. Although, AplnR and its recently identified ligand, apelin, show high levels of mRNA expression in the heart, their functional significance in the cardiovascular system is not yet fully understood [18,21]. Apelin is synthesized as a 77-amino acid peptide processed into various C-terminal fragments, including: apelin-36, apelin-19, apelin-17, apelin-13, and [Pyr1]-apelin-13 [22,23]. [Pyr1]-apelin-13 has been recognized as the predominant isoform of apelin in human plasma and cardiac tissue [22,24–26].

Extensive clinical use of [Pyr1]-apelin-13 has been seriously hampered due to the unstable nature of the peptide in both in vitro and in vivo conditions [43,44]. The plasma instability of apelin is due to the rapid degradation of the peptide [22,25,26,45] which results in significantly short plasma half-life (<8 min) [46]. Nanocarrier drug formulations have been found beneficial for the sustained delivery of therapeutic compounds in various cardiac disease [27–29]. Among a variety of nanocarriers, liposomes are widely considered as suitable vesicles for potential targeted drug delivery [30–32]. In particular, liposomal peptide delivery platform caters to various biological needs [33–35]. To promote effective drug targeting, ligands for specific cell surface receptors need to be incorporated in the nanocarriers [36]. Liposomes encapsulating drugs can perform this function via utilizing a variety of loading methods [37–39]. Considering the fact that surface modifications of liposomes can help to improve the drug release properties of these carriers, a considerable amount of research has been focused on the liposome-drug interactions [40–42].

In this study, a novel liposomal nanocarrier system, incorporated with polyethylene glycol (PEG) polymer on the surface, was utilized in order to deliver [Pyr1]-apelin-13 as a therapeutic molecule into the injury site in a TAC mouse model. The effect of sustained release of [Pyr1]-apelin-13 on the hypertrophic response of the heart was assessed.

2. Materials and methods

2.1. Liposome preparation and [Pyr1]-apelin-13 encapsulation

Lipids used in the preparation of liposomes include 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino (polyethylene glycol)-3400] (DSPE-PEG(3400)- NH2) (Laysan Bio, Inc.), cholesterol (Sigma), and 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) (Avanti polar lipids, Inc.). PEG-containing liposome (lipoPEG) was prepared by dissolving cholesterol (5 mg), NH2-PEG-DSPE (2 mg), and DSPC (10 mg) in 1 mL of chloroform in a round-bottom (RB) flask. The mixture was evaporated in a rotary evaporator under vacuum to make a thin layer of molecules in the RB flask. The lipid layer was hydrated by the addition of 1 mL double distilled water sonicated for 30 min to obtain a turbid liposome solution. The solution was frozen and lyophilized to obtain solvent-free dry liposomes. [Pyr1]-apelin-13 was encapsulated by hydrating the liposomes (17 mg) with 3.3 mg of pyroglutamyl [Pyr1]-apelin-13 in 1 mL double distilled water followed by 30 min sonication. In order to get the maximum encapsulation, the mixture was frozen and lyophilized again followed by rehydrating it with 1 mL double distilled water and sonicated before use. Hereby, liposome-PEG-[Pyr1]-apelin-13 (lipoPEG-PA13) was prepared.

2.2. Atomic force microscopy

Samples were prepared for atomic force microscopy (AFM) by drop-casting and drying under vacuum of 10 μL droplets with a liposome concentration of 0.01 mg/mL on the surface of clean silicon wafers. AFM imaging was performed with Park Systems NX10 (Suwon, Korea) AFM instrument. Samples were imaged in semi-contact mode with standard commercial cantilevers (k = 5–9 N/m, R < 10 nm), at 1 Hz scan speed and ~30% oscillation damping.

2.3. Dynamic light scattering measurements

Solutions were prepared from dry lyophilized liposomes at a concentration of 1 mg/mL. Samples were vortexed until no visible sediment could be detected. Dynamic Light Scattering (DLS) measurements were performed using Brookhaven 90 plus DLS nanosizer (Brookhaven Instruments Corporation, Holtsville, NY). The autocorrelation function was approximated manually with a single-relaxation process using second-order term for the cumulant analysis of particle polydispersity.

2.4. In vitro [Pyr1]-apelin-13 release study

In vitro release of [Pyr1]-apelin-13 was measured using rapid equilibrium dialysis (RED) plate assay (Thermo Scientific Co.). The plate is composed of disposable high-density polypropylene and equilibrium dialysis membrane inserts compartmentalized in to buffer and sample chambers. Each insert is comprised of two sideby- side chambers separated by an O-ring-sealed vertical cylinder of dialysis membrane with molecular weight cutoffs (12 kDa). Stock solution of liposome-PEG-[Pyr1]-apelin-13 (lipoPEG-PA13) was prepared in 2 mL PBS containing 100 nM concentration of [Pyr1]-apelin-13 peptide. The lipoPEG-PA13 sample (750 μL) and the buffer PBS (500 μL) were placed into the corresponding sample (donor) and buffer chambers, respectively. The entire unit was then covered with sealing tape and incubated at 37 °C on an orbital shaker at approximately 300 rpm. At each time point of analysis, a volume of 10 μL was withdrawn from the buffer side of the chamber and replaced with the same volume in return. At 0 h (start point) and 24 h (end point), a volume of 10 μL was collected from both the sample (donor) and the buffer chambers. Despite the long duration of incubation time (24 h), there was no increase in the sample volume due to the hydrostatic pressure. All drug release experiments were done with samples in triplicates. The vials containing collected sample (donor) and buffer solutions were stored at 4 °C before mass spec analysis.

2.5. Liquid chromatography/mass spectrometry analysis

The concentration of [Pyr1]-apelin-13 released from liposome was measured using liquid chromatography (LC, Shimadzu-LC-20AB Prominence Liquid Chromatogram) coupled with a mass spectrometry (MS) device (4000 Q Trap). Briefly, the collected 10 μL volume of either buffer or donor was added to 20 μL water and 30 μL methanol to precipitate the peptide. Chromatographic separation was performed on a C18 reversed-phase column (XBridge C18, 3.0 × 150 mm, 3.5 μm particle size) by injecting 10 μL per sample. Gradient elution was employed at a flow rate of 0.3 mL/min with the following solvent and conditions: 0–0.5 min 20% acetonitrile/0.1% formic acid, water/0.1% formic acid/5 mM ammonium acetate, 0.5–7 min gradient from 20% to 90%; 7–8 min 90%; 8–12 min 20%. Based on our established protocol, mass spectrometric detection was conducted specifically at the Q1 (512.060 mass in Da) and Q3 (263.000 mass in Da) regions accomplished with an electrospray ionization (ESI) source in positive ion mode at 5.5 kV spray voltage. Data acquisition was carried out by the AB Sciex Analyst version 1.6.1 software. The concentration of [Pyr1]-apelin-13 in the sample chamber and those released in to the buffer chamber were measured from the calculated peak areas relative to the internal standard.

2.6. In vitro assessment of [Pyr1]-apelin-13 effect on APJ translocation

The influence of [Pyr1]-apelin-13 formulation (lipoPEG-PA13) – versus commercially available [Pyr1]-apelin-13 peptide – on the APJ translocation efficacy was tested in vitro, using human embryonic kidney (HEK) 293 cells overexpressing GFP-APJ as described elsewhere [47]. HEK cells, transfected with human APJ, were cultured in DMEM containing 1% penicillin/streptomycin and 10% FBS. Following 60 min incubation with lipoPEG-PA13, the GFP-APJ fluorescence was tracked using a high-resolution confocal laser scanning microscope (Zeiss LSM510 Meta).

2.7. Mouse model of TAC – [Pyr1]-apelin-13 nanocarriers administration

All procedures involving animal use, housing, and surgeries were approved by the Stanford Institutional Animal Care and Use Committee (IACUC). Animal care and interventions were provided in accordance with the Laboratory AnimalWelfare Act. Male 10–13 weeks old C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and divided randomly into five groups: I and II) sham controls with no aortic constriction, treated with either saline or [Pyr1]-apelin-13 formulation, III to V) mice with TAC injury treated with either saline, regular [Pyr1]-apelin-13, or lipoPEG-PA13 (n > 4, Fig. 1A–D).

Fig. 1.

Schematic representation (top) and AFM images (bottom) demonstrating the structure of empty PEG liposomes (A and B, respectively), and PEG liposomes encapsulated with [Pyr1]-apelin-13 (C and D, respectively). Panels B and D demonstrate the morphology of the nanocarrier systems used in this study.

For constriction of transverse thoracic aorta [8], mice were anesthetized using an isoflurane inhalational chamber, followed by endotracheal intubation using a 22-gauge angiocatheter (Becton, Dickinson Inc., Sandy, Utah) connected to a small animal ventilator (Harvard Apparatus, Holliston, MA). A left thoracotomy was performed via the fourth intercostal space, the transverse aorta was identified, and a 6.0 silk suture was placed between the innominate and left carotid arteries. The suture was tightened around a 27^1/2 gauge blunt needle, placed parallel to the aorta (Fig. 1B) to induce a constriction of ~0.4 mm in diameter. In sham control mice, an identical procedure was conducted, except for the constriction of the aorta. Mice received [Pyr1]-apelin-13 or saline (control) intraperitoneally (300 μg/kg body weight) at days 1 and 7 post-surgery.

2.8. Echocardiography

In vivo heart function was evaluated by echocardiography at 1 day prior to surgery (baseline) and on days 1, 7, and 14 post-surgery (Fig. 1A). Two-dimensional (2D) analysis was performed using a GE Vivid 7 ultrasound platform (GE Health Care, Milwaukee, WI) equipped with a 13 MHz transducer. Mice were anesthetized with isoflurane (2% inhalation). 2D clips and M-mode images were obtained in the short axis view from the mid-LV at the tips of the papillary muscles. Fractional shortening (FS), LV internal diameter at end diastole (LVIDd) and systole (LVIDs), and posterior LV wall thickness at end diastole (LVPWd) were measured. A minimum number (n) of 4 mice per study group was used for the echo evaluations. Measurements were performed by two independent observers blinded to study groups.

2.9. Measurement of [Pyr1]-apelin-13 concentration in blood plasma

24 h post-surgery, sham and TAC operated mice were injected intraperitoneally with either saline or the [Pyr1]-apelin-13 nanocarriers (lipoPEG-PA13) at 0.4 mg/kg body weight. Wild type mice without surgery and with no injections were used as controls to measure the apelin-13 baseline levels. At days 1, 4, and 6 post injection, mice were anesthetized with isoflurane (100 mg/kg, inhalation) and blood was collected using two different techniques: 1) from the Orbital Sinus (Retro-orbital technique) by inserting the tip of a microhematocrit blood tube into the corner of the eye socket, underneath the eyeball, and directing the tip at towards the middle of the eye socket at a 45-degree angle (details in Ref. [48]). 2) from the tail vein; using a straight edge razor, approximately 1 cm of the tail was quickly removed. For additional samples at later time point, blood sample was obtained by removing ~2–3 mm of additional tail (details in Ref. [48]). The blood obtained from either approaches was placed in a polypropylene tube containing 0.1 volume of 3.8% sodium citrate, pH 7.4. Plasma was then prepared by centrifuging the samples at 15,000 g for 10 min (at 4 °C) using a table-top microcentrifuge (Eppendorf 5424R). Aliquots were stored at −80 °C before analysis.[Pyr1]-apelin-13 concentrations were determined by commercial enzyme linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions (Apelin EIA kit, Phoenix Pharmaceuticals, Burlingame, CA). Each assay was performed in duplicates. Standard curve was fitted using the 4-parameter logistic (4 PL) nonlinear regression model [47].

2.10. Histological analysis

Histological analysis was performed following standard protocols for paraffin embedding. Mounted heart samples were stained for Masson’s trichrome (TC, to label muscle fibers in red, collagen in blue, cytoplasm in pink, and nuclei in dark brown). A minimum of 4 sections per sample were used.

2.11. Statistical analysis

The number of samples (n) used in each experiment is noted in the text. Dependent variables are expressed as means ± SEM, unless noted otherwise. The differences in the means were tested using ANOVA and Student T-test to check for statistical significance (P < 0.05).

3. Results

3.1. AFM characterization of liposomes and liposome encapsulating [Pyr1]-apelin-13

AFM imaging of the particles, performed in tapping mode, generated the topography maps of the PEG liposomes (Fig. 1). Both the empty liposomes and PEG liposomes encapsulating [Pyr1]-apelin-13 (Fig. 1A and C, respectively) exhibited semi-spherical structure with relatively narrow size distribution. The lipoPEG-PA13 nanoparticles formed objects akin to “sunny side up” fried eggs. There was only a slight difference in the morphology and height of the particles in empty versus apelin-13-loaded conditions (Fig. 1B and D, respectively).

3.2. Particle size characterization using dynamic light scattering

Dynamic light scattering (DLS) was used to analyze the size distribution of the empty lipoPEG particles and the particles laden with [Pyr1]-apelin-13, in the absence or presence of 0.5% BSA (Fig. 2A). The average size of the lipoPEG alone was estimated to be 0.57 ± 0.02 (SD) μm. Upon inclusion of 0.5% BSA, there was almost no change in size (0.53 ± 0.02 (SD) μm) for lipoPEG particles. However, [Pyr1]-apelin-13 encapsulation in lipoPEG showed a striking reduction in particle size to 0.06 ± 0.00 μm. Inclusion of 0.5% BSA to particles with encapsulated [Pyr1]-apelin-13 increased their size to 0.22 ± 0.02 (SD) μm.

Fig. 2.

A: DLS data demonstrating the liposome particle sizes. Error bars indicate standard deviations of liposome sizes based on cumulant analysis. B and C: time course analysis of [Pyr1]-apelin-13 release from liposomes in the absence (B) and presence (C) of %0.5 BSA, by rapid equilibrium dialysis plate assay. The quantity (μg/mL) of [Pyr1]-apelin-13 released from donor to buffer reservoir over a 24 h time course at various intervals is indicated.

3.3. Liposomal encapsulation promotes sustained in vitro release of [Pyr1]-apelin-13

[Pyr1]-apelin-13 encapsulation in lipoPEG particles (lipoPEG-PA13) resulted in sustained and extended drug release under physiological conditions in vitro when monitored over a 24 h period at different time intervals (Fig. 2B and C). The peak [Pyr1]-apelin-13 release profile was obtained between 15 and 20 h period followed by a saturation curve at the end of 24 h. [Pyr1]-apelin-13 release from lipoPEG-PA13 was accelerated (18.6 μg/mL) in the presence of 0.5% BSA (Fig. 2C).

3.4. LipoPEG-PA13 nanocarriers promote APJ translocation in vitro

The in vitro effect of [Pyr1]-apelin-13 nanocarriers on APJ translocation was analyzed in APJ-GFP⁺ HEK293 cells and compared to that induced by commercially available [Pyr1]-apelin-13 (300 nM in culture media [47], Fig. 3). Tracing the fluorescent APJ receptors using high-resolution confocal microscopy demonstrated that lipoPEG-PA13 nanocarriers stimulated APJ translocation from the cell membrane towards the cytoplasm more efficiently than that caused by the regular [Pyr1]-apelin-13. The lipoPEG-PA13 nanocarriers also exhibited a qualitatively higher GFP signal after 60 min (Fig. 3).

Fig. 3.

In vitro assessment of the effect of the [Pyr1]-apelin-13 nanocarriers on the APJ (green) translocation (white arrows) in HEK APJ-GFP + cells (t = 60 min). (A) and (B): negative controls, without [Pyr1]-apelin-13. (C) and (D): regular [Pyr1]-apelin-13 effect. (E) and (F): the effect of addition of lipoPEG-[Pyr1]-apelin-13 nanocarriers. DAPI staining the nuclei (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.5. Treatment with lipoPEG-PA13 nanocarriers attenuates TAC-induced cardiac dysfunction in vivo

The lipoPEG-PA13 was administered in vivo in TAC operated mice and sham-operated controls by means of single weekly injections for two weeks (Fig. 4). Serial echocardiography at baseline and days 1, 8, and 15 after injury demonstrated significant beneficial effects of lipoPEG-PA13 administration in inhibiting pressure overload-induced LV dysfunction (Fig. 5). No significant differences were observed in echo parameters of the three TAC groups at day 1 post-surgery (Fig. 5A). In saline-treated animals (TAC + saline), TAC resulted in LV hypertrophic growth, chamber dilation, and a significant drop in cardiac function at days 8 and 15 post operation (Fig. 5B and C, respectively). In contrast, the application of [Pyr1]-apelin-13 nanocarriers (TAC + lipoPEG-PA13) blunted LV dilation at both end-diastole and systole (LVIDd and LVIDs, respectively), attenuated the increase in posterior wall thickness (LVPWd) at day 15, and preserved cardiac contractility (FS) (Fig. 5B–D). In contrast, the administration of commercially available [Pyr1]-apelin-13 (TAC + [Pyr1]-apelin-13) had a more modest effect in diminishing the hypertrophic response to pressure overload. Sham operated groups injected with either saline (data now shown for the simplicity of the graphs) or lipoPEG-PA13 showed no significant changes in cardiac dimensions or function. Thus, lipoPEG-PA13 attenuated pressure overload-induced cardiac dysfunction up to two weeks post injury.

Fig. 4.

Schematic representation of the experimental procedure used to assess the effect of lipoPEG-[Pyr1]-apelin-13 nanocarriers administration in vivo, on the TAC-induced cardiac hypertrophy in mouse.

Fig. 5.

Percentage changes in echocardiographic parameters compared to baseline (divided to the pre-surgery values), at days 1 (A), 8 (B), and 15 (C) post-surgery (TAC). Moreover, panel (D) demonstrates the absolute fractional shortening (FS) values for the four different groups as a function of time post-surgery. Saline (sham controls), [Pyr1]-apelin-13, or lipoPEG-[Pyr1]-apelin-13 (lipoPEG-PA13) were injected IP (300 μg/Kg body weight) at days 1 and 7 post-surgery. While sham controls, either injected with saline (not shown) or lipoPEG-PA13 nanocarriers , showed no significant changes in the heart function over 2-week time period, TAC hearts with saline treatment showed significant decline in cardiac contractility (fractional shortening, FS) and an increase in LV diameter (LVID) as well as LV posterior wall thickness (LVPW), indicating a hypertrophic response and cardiac dysfunction. Administration of commercial [Pyr1]-apelin-13 resulted in slight inhibition of cardiac remodeling and increase in FS, when compared with TAC controls. The greater effect however, was observed in the group treated with lipoPEG-PA13 carriers in which cardiac contractility (FS) was significantly improved compared with those in other TAC groups (D), and hypertrophic remodeling was significantly inhibited. As shown in panel (D), the four animal groups of this study showed varying levels of FS at the baseline. Considering these differences, the significant effect of nanocarriers treatment in improving FS is evident. A minimum of four replicates were analyzed for each group. Statistical analysis was performed using one-way ANOVA test. Values are reported as average ± SEM. *: P < 0.05 compared to sham controls ( ). ▲: P < 0.05 compared to TAC injected with [Pyr1]-apelin-13 ( ). E: Representative heart sections with Masson’s trichrome staining for all experimental groups. While TAC caused a remarkable left ventricular hypertrophic growth along with a significant amount of fibrotic tissue in untreated TAC hearts, administration of [Pyr1]-apelin-13 or lipoPEG-PA13 nanocarriers resulted in remarkable inhibition of the LV growth, attenuating TAC-induced cardiac dysfunction, and diminishing fibrosis. Scale bar is 2 mm.

In order to further assess the effect of [Pyr1]-apelin-13 nanocarriers on heart tissue structure and remodeling, mice were sacrificed two weeks post injury and hearts were analyzed. Histology (trichrome staining) of heart sections demonstrated no significant changes in cardiac morphology of sham controls, either in saline or [Pyr1]-apelin-13 treated animals (Fig. 5E). TAC + saline-treated mice showed a remarkable increase in the size of LV and the amount of fibrotic tissue. TAC mice treated with lipoPEG-PA13 showed reductions in both the size of the LV and the extent of cardiac fibrosis, when compared to TAC + saline and TAC mice treated with regular [Pyr1]-apelin-13 (Fig. 5E).

3.6. Liposomal encapsulation promotes sustained release of [Pyr1]-apelin-13 in vivo

IP administration of lipoPEG-PA13 in both sham-operated and TAC mice resulted in significantly elevated levels of [Pyr1]-apelin-13 in blood plasma at days 1, 4, and 6 post injection, when compared to those in control animals (injected with saline, or not injected, WT) (Fig. 6A and B). The peak [Pyr1]-apelin-13 levels in blood plasma in the TAC and sham mice were obtained at days 1 and 4 post injection, respectively. Moreover, in comparison between sham ( ) and TAC ( ) mice injected with lipoPEG-PA13, [Pyr1]-apelin-13 concentration in TAC mice was significantly greater than that in sham group at each time point (except day 4).

Fig. 6.

[Pyr1]-apelin-13 release in vivo; using ELISA, the [Pyr1]-apelin-13 concentration in blood plasma was measured at 1, 4, and 6 days post IP injection, in A: Sham mice injected with saline (control ) or lipoPEG-[Pyr1]-apelin-13 (lipoPEG-PA13) ( ), and B: TAC mice injected with saline (control ) or lipoPEG-PA13 nanocarriers ( ). These were compared with [Pyr1]-apelin-13 concentration in blood plasma of wild type (WT) mice with no injury and no injections ( A and B). In both sham and TAC animals, using the nanocarriers resulted in significantly elevated levels of [Pyr1]-apelin-13 in blood plasma up to 6 days post injection, compared to those in saline-injected animals as well as those in control WT mice. A minimum of four replicates were analyzed for each group. Statistical analysis was performed using one-way ANOVA test. Values are reported as average ± SEM. +: P < 0.05 compared to WT controls. *: P < 0.05 compared to same time, injected with saline. ■: P < 0.05 compared to sham at the same time, injected with lipoPEG-PA13 carriers.

4. Discussion

Recent studies have shown that the loss of apelin function, together with deficiency of its receptor APJ, correlated with cardiovascular disease [20,49,50]. Some studies have consistently suggested that decreased plasma or myocardial levels of apelin/APJ levels can lead to the incidence of various cardiac defects [14,51,52]. To date, a significant amount of research has evaluated the role of apelin administration in regulating cardiac dysfunction in various models of heart disease, introducing this peptide as an attractive target for pharmacotherapy studies in the setting of heart failure [18,21,50,44]. Among other beneficial effects, apelin delivery using osmotic pumps has been shown to prevent aortic aneurism [53], ameliorate myocardial reperfusion injury [54], induce vasodilatation [46], and prevent deleterious hypertrophy remodeling [47]. However, the success of conventional systemic drug administration approaches is limited due to several factors including the lack of targeting capability into a pathological site, poor control of sustained drug delivery during the desired therapeutic time, the necessity of a high dose at the target site, nonspecific toxicity, and peptide instability [55–57]. Here, we investigated the use of a liposome nanocarrier system for sustained delivery of exogenous [Pyr1]-apelin-13 and its effect on TAC-induced cardiac hypertrophy in the mouse model.

In order to increase the stability of the nano-liposome in the circulation, the absorbance of the opsonin-based proteins at the surface of nanoparticles should be prevented [58]. One of the well-recognized approaches for enhancing stability of nano-size materials in circulation is to coat their surfaces by polyethylene glycol (PEG) polymers [59]. PEG polymer has been used extensively for the covalent alteration of biological macromolecules and surfaces for various biomedical and pharmaceutical applications [60,61]. This polymeric shell can prevent plasma’s opsonin proteins interactions with the surface of nanocarriers and hence rescue their removal by reticuloendothelial system [30,62–64]. PEGylation process increases the molecular mass of protein complexes and shields them against proteolytic enzymes, hence, prolongs their circulation half-life [60,65]. Thereupon, a lower number of injections or a less chronic drug administration will be required to maintain plasma concentrations of the PEGylated proteins or peptides [66]. In this study, PEG polymer was incorporated onto the surface of the liposome carriers in order to improve blood plasma stability during in vitro and in vivo experiments.

AFM imaging of the nanocarriers demonstrated the integration of [Pyr1]-apelin-13 into the liposomal particles, with the formation of a mostly homogeneous structure without a clearly defined core and shell (Fig. 1). If we approximate the shape of the two particles in Fig. 1A with a half of an oblate ellipsoid, the volume would be:

$$V_{\frac{1}{2}\text{oblate}\text{ellipsoid}}=\frac{1}{2}·\frac{4}{3}\mathrm{\pi }{a}^{2}b$$ (1)

where a and b are halves of long and short axes of the ellipsoid, respectively. The same particles would take a shape of a sphere in solution, the diameter of which would be:

$$D=2·{\left(\frac{V}{\raisebox{1ex}{$4$}\!\left/ \!\raisebox{-1ex}{$3$}\right.\mathrm{\pi }}\right)}^{⅓}$$ (2)

The diameters, obtained in such a fashion are about 0.5 μm, which is in close agreement with the DLS data (Fig. 2A). Using this approximation, the typical volume and, therefore, the solution size of BSA-coated liposomes were calculated to be about the same.

The DLS characterization of plain liposomes (lipoPEG) and those encapsulated with [Pyr1]-apelin-13 demonstrated relatively high monodispersity of all sets of particles which was not significantly affected by the addition of BSA (Fig. 2A). Interestingly, it appears that the addition of BSA drives the particle size to 0.5 μm, which can be related to the general stability of a BSA-coated particle in a water solution. The relatively large size of particles contributes to the extension of the release period, since it takes longer for the particles to get incorporated through the outer skin layers.

Considering the fact that sustained drug release is an important pharmacokinetic entity, the effect of lipoPEG (14 μg/mL) particles in sustained and prolonged in vitro release of [Pyr1]-apelin-13 over a 24 h time course was quite significant (Fig. 2B and C). [Pyr1]-apelin-13 release from lipoPEG-PA13 carriers was accelerated (18.6 μg/mL) in the presence of 0.5% BSA (Fig. 2C) suggesting that BSA had no effect in promoting the drug sustainability. Several studies have reported efficient targeting of protein or peptide based drugs via PEG liposome encapsulation [67–69]. Of particular note, PEGylated DSPE liposomes have shown great potential in promoting the sustained release of several peptide based drugs in vitro [70–72].

Enhanced in vitro efficacy of [Pyr1]-apelin-13 via nanoencapsulation was also demonstrated by no adverse effect on cell viability, and significantly increased APJ translocation in the HEK cells when compared to that induced by [Pyr1]-apelin-13 alone (Fig. 3, Suppl. Figs. 1 and 2). The cationic PEGylated Liposomes are recognized for their intricate ability to become incorporated into pre-formed vesicles, hence, improving cellular binding in vitro [73]. Cellular uptake of peptide-encapsulating PEGylated liposomes has been reported to take place through clathrin-mediated endocytosis pathway [74]. In our future study, we aim to elucidate the precise cellular binding and uptake mechanisms that are involved in these nanocarriers–cell interactions.

Administration of lipoPEG-PA13 nanocarriers after TAC demonstrated a significant benefit of the drug in attenuating pressure overload-induced cardiac dysfunction (Fig. 5). On day 8, the administration of lipoPEG nanocarriers resulted in significantly reduced LV dimensions (P < 0.05) when compared with those in regular [Pyr1]-apelin-13-treated mice, while there was no significant difference in LV contractility (FS) (Fig. 5B). This suggests that at earlier time points, lipoPEG-PA13 administration effectively interfered with cardiac hypertrophic remodeling. At week 2, in contrast to the TAC mice treated with saline or regular [Pyr1]-apelin-13 (without carriers), administration of the lipoPEG-PA13 resulted in a significant improvement in LV contractility (FS) (P < 0.05). We have previously demonstrated that naked [Pyr1]-apelin-13 administration required continuous infusion through osmotic pump to achieve a comparable effect [47]. Here we found that the lipoPEG-PA13 formulation, administered only once weekly, was capable of attenuating cardiac hypertrophy and the resulting dysfunction for up to two weeks post injury. It should be noted that the four animal groups studied showed varying levels of absolute FS at baseline (Fig. 5D); thus, we utilized percent change from baseline to correct for these differences (Fig. 5A–C). Hence, although the absolute FS of the TAC + apelin group was above that of the TAC + nanocarriers group at several time points in Fig. 5D, comparatively, the percentage improvement in contractility was significantly greater in the nanocarrier group, as demonstrated in Fig. 5C. Histology of the mouse hearts treated with lipoPEG-PA13 nanocarriers confirmed the in vivo effect of [Pyr1]-apelin-13 nanocarriers in diminishing fibrosis and cardiac hypertrophy two weeks post injury (Fig. 5E).

The in vivo drug release study performed on the TAC, sham-operated, and wild type (with no surgery) mice revealed the significant effect of the lipoPEG nano-capsulation in prolonging the apelin-13 lifetime in the blood plasma for up to 6 days (Fig. 6). This is while regular commercial apelin appears rapidly clears from the blood circulation, with a drastically short plasma half-life (no longer than 8 min) [46]. Remarkably, the effect of nanocarriers in sustained [Pyr1]-apelin-13 release was more significant in the TAC mice, in comparison with that in sham controls. This can be related to the induced changes in plasma proteins due to the acute cardiac (TAC) injury and/or the stress caused by surgery (known as the acute phase response) [75–79]. It is well recognized that the surface of nanocarriers gets covered by various proteins upon their entrance into the biological fluid (e.g., blood [58,80]). The type, amount, and thickness of the associated proteins on the surface of nanocarriers (so called “protein corona”) can define their biological fate [81]. Since the protein source determines the type and composition of associated proteins on the surface of these carriers [82], the variation of plasma proteins in TAC-operated mice may change the protein corona thickness and composition, hence, leading to a prolonged blood circulation time. Further assessment of the potential role of protein corona formation on the drug release from nanocarriers would be of interest for the future investigations.

5. Conclusions

In summary, we have developed an engineered nanocarrier system, consisting of liposomal nanocarriers incorporating PEG polymer, that demonstrated significantly enhanced efficacy compared to the non-encapsulated, commercially available [Pyr1]-apelin-13, in the delivery and sustained release of drug both in vitro and in vivo. Administration of [Pyr1]-apelin-13 nanocarriers significantly attenuated left ventricular hypertrophy and pressure overload-induced cardiac dysfunction in the mouse model of TAC. An increase in the particles polydispersity could be a further beneficial way to prolong the release. Testing release characteristics of a highly-polydisperse particle formulation would be of interest for the future studies. Further investigations on other therapeutic factors that can be delivered using these carriers, and modification and/or optimization of these devices can establish a new generation of drug delivery systems with unprecedented efficacy to treat various heart injuries and diseases.

Abbreviations

LipoPEG-PA13

liposome-PEG-[Pyr1]-apelin-13

PEG

polyethylene glycol

TAC

transverse aortic constriction

LV

left ventricle

LVIDd

left ventricle internal diameter at end diastole

LVIDs

left ventricle internal diameter at end systole

LVPWd

left ventricle posterior wall thickness at end diastole

LVPWs

left ventricle posterior wall thickness at end systole

FS

fractional shortening

FM

atomic force microscopy

HEK

human embryonic kidney

APP

acute phase protein

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2014.08.045.