Abstract
This article reports a promising approach to enhance the oral delivery of nuciferine (NUC), improve its aqueous solubility and bioavailability, and allow its controlled release as well as inhibiting lipid accumulation. NUC-loaded poly lactic-co-glycolic acid nanoparticles (NUC-PLGA-NPs) were prepared according to a solid/oil/water (s/o/w) emulsion technique due to the water-insolubility of NUC. PLGA exhibited excellent loading capacity for NUC with adjustable dosing ratios. The drug loading and encapsulation efficiency of optimized formulation were 8.89 ± 0.71 and 88.54 ± 7.08%, respectively. NUC-PLGA-NPs exhibited a spherical morphology with average size of 150.83 ± 5.72 nm and negative charge of −22.73 ± 1.63 mV, which are suitable for oral administration. A sustained NUC released from NUC-PLGA-NPs with an initial exponential release owing to the surface associated drug followed by a slower release of NUC, which was entrapped in the core. In addition, ∼77 ± 6.67% was released in simulating intestinal juice, while only about 45.95 ± 5.2% in simulating gastric juice. NUC-PLGA-NPs are more efficient against oleic acid (OA)-induced hepatic steatosis in HepG2 cells when compared to naked NUC (n-NUC, *p < 0.05). The oral bioavailability of NUC-PLGA-NPs group was significantly higher (**p < 0.01) and a significantly decreased serum levels of total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), as well as a higher concentration of high-density lipoprotein cholesterol (HDL-C) was observed, compared with that of n-NUC treated group. These findings suggest that NUC-PLGA-NPs hold great promise for sustained and controlled drug delivery with improved bioavailability to alleviating lipogenesis.
Keywords:
Introduction
The excess lipid accumulation within hepatocytes followed by subsequent inflammation was eventually developed into liver damage (Woods et al., Citation2015). Several alkaloid extractions of lotus leaf, a traditional Chinese medicinal herb, have been shown to possess therapeutic potentials for obesity and atherosclerosis via accelerating lipid metabolism, reducing triglyceride accumulation in adipocytes and inhibiting the absorption of lipids and carbohydrates (Wang et al., Citation2015). Nuciferine (NUC), an aporphine alkaloid, is thought to be responsible for the active ingredient of Nelumbo nucifera, which possessed a broad range of pharmacological activities containing ameliorating hyperlipidemia, lowering cholesterols, dilating vessels, stimulating insulin secretion and improving hepatic lipid metabolism. In our previous study, we successfully demonstrated that NUC owns potential of attenuating or inhibiting lipid accumulation and inflammation and its possible underlying mechanism (Zhang et al., Citation2015).
The clinical application of NUC has been hampered due to its bioavailability. We have calculated the values of absolute bioavailability according to oral doses of 2.0, 5.0 and 10.0 mg/kg and intravenous (IV) administration of 0.2 mg/kg NUC in rats. The results were (3.8 ± 1.4), (4.2 ± 1.3) and (3.9 ± 1.0)%, respectively (Gu et al., Citation2014). We concluded the low absolute bioavailability was related to poor absorption, rapid metabolism and rapid systemic elimination.
Oral administration was suggested to own the maximum patient compliance, while the poor bioavailability is a conundrum not yet solved (Pathak & Raghuvanshi, Citation2015). In order to reach therapeutic level, drugs with poor oral bioavailability are administered at increased dose or designed as a novel delivery system that can exhibit improved pharmacokinetic profiles. Increased dose may induce the poor compliance, wastage of drug, which is not economical bearing, especially the expensive drugs and most importantly the adverse effects. Pharmaceutical manufacturers thus optimize the drug molecule, which has gradually evolved into the progress of micro-sized and nano-sized medication (Cherniakov et al., Citation2015).
Nanomedicine is rapidly gaining recognition for enhancing the bioavailability of drugs in their adjustable dosage formulations, especially highly hydrophilic drugs (Ozeki & Tagami, Citation2013). Poly (lactic-co-glycolic acid) (PLGA) is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA) that has been approved for clinical application by the US Food and Drug Administration (FDA) (Zhao et al., Citation2016). PLGA has excellent biocompatibility and degradability in physiological environments and the biodegradation products (lactic acid and glycolic acid) are naturally occurring metabolites. By virtue of these advantages, PLGA has been used as an efficient carrier for food and drug delivery. Shi et al. employed mono-PEGylation combined with PLGA delivering Radix Ophiopogonis polysaccharide, a poorly water-soluble macromolecule, to solve the problem of its relatively short half-life in vivo (Shi et al., Citation2014). Chen et al. developed a biodegradable micellar system composed by glycyrrhetinic acid and PLGA to encapsulate Tanshinone IIA targeting hepatocellular carcinoma (Chen et al., Citation2016).
In this study, we perfected and fine-tuned the bioavailability of drug by loading water insoluble NUC in PLGA based on a solid/oil/water (s/o/w) emulsion technique. The NUC-loaded poly lactic-co-glycolic acid nanoparticles (NUC-PLGA-NPs) were characterized with regard to particle size, zeta potential, morphology and encapsulation efficiency and drug loading as well as the in vitro release of NUC. Both the effects of n-NUC and NUC-PLGA-NPs against OA-induced hepatic steatosis in HepG2 were studied. NUC’s oral bioavailability and lipid lowering in vivo were also evaluated in male Sprague Dawley (SD) rats.
Materials and methods
Materials
Poly (D,L-lactic-co-glycolic acid) (PLGA, molar ratio of D,L-lactic to glycolic acid, 50:50, MW = 30 KDa) was purchased from Jinan Daigang Biomaterial Co., Ltd (Shandong, China); bovine serum albumin (BSA) was acquired from Amresco (Solon, OH); NUC, DIL, coumarin-6, Oil-red-O (ORO), oleic acid (OA), and vitamin E were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin solution (5 KU/mL) were purchased from Thermo Fisher Scientific (Waltham, MA). A Cell Counting Kit-8 (CCK8) was obtained from Dojindo Molecular Technologies Inc. (Tokyo, Japan). Annexin V-PE/7AAD Apoptosis Detection Kit was obtained from BD Pharmingen (San Diego, CA). All other chemicals used were analytical grade and organic solvents were high performance liquid chromatography (HPLC) grade.
Cell culture
Human hepatocellular carcinoma (HCC) cells HepG2 was purchased from Cell Culture Center of Shanghai Institutes of the Chinese Academy of Sciences (Shanghai, China). HepG2 were cultured in high glucose DMEM containing 10% FBS and 1% penicillin–streptomycin under an incubator with 5% CO2 at 37 °C.
Animals
Adult male SD rats weighing 220–240 g were purchased from SLAC Laboratory Animal Co. Ltd (Shanghai, China). Rats were maintained at stable temperature (23 ± 2 °C) and humidity (45–55%) under a 12-h light/dark cycle. All animal experiments were in accordance with the Animal Care and Use of Laboratory Animals of Shanghai General Hospital and followed the guidelines of the Animal Welfare Act.
Preparation of NUC-PLGA-NPs
NUC-PLGA-NPs were prepared according to a s/o/w emulsion technique with moderate modification (Perez et al., Citation2002). Briefly, PLGA was dissolved in dichloromethane and acetone at a volume ratio of 3:2 to acquire a uniform PLGA solution (10 mg/mL). Free NUC was added to the PLGA solution and sonicated at 100 W for 60 s to produce the s/o primary emulsion. The acquired emulsion was then added to 4 mL of BSA solution (1% w/v) and again sonicated at 100 W for 30 s twice time to generate the final s/o/w emulsion. To disperse the final s/o/w emulsion, 30 mL of distilled water was added followed by 4 h of magnetic stirring for the removal of residual organic solvents. The NPs were finally acquired after centrifugation at 14 000 rpm for 30 min and the supernatant was discarded. The obtained NUC-PLGA-NPs were washed with distilled water three times before lyophilization with vacuum freeze dryer (VirTis, Stone Ridge, NY) under −50 °C. The freeze-dried powder was stored at 4 °C for future use.
Characterization of NUC-PLGA-NPs
Encapsulation efficiency and drug loading
To evaluate the encapsulation efficiency ratio (EE%) and drug loading ratio (DL%), NUC-PLGA-NPs with various amounts of NUC were prepared as described in the part of preparation of NUC-PLGA-NPs. NUC levels in the PLGA nanoparticles were assayed by HPLC. The EE% of the NUC-PLGA-NPs was calculated as NUC encapsulated in respect to the feeding NUC (wt%/wt%) to prepare the NUC-PLGA-NPs and the DL% was expressed as the amount of NUC encapsulated in respect to the NUC-PLGA-NPs (wt%/wt%). To disrupt NPs structure, 5 mg lyophilized NUC-PLGA-NPs were dissolved in 1 mL methanol and vortexed with ultrasonic waves for 10 min to ensure encapsulated NUC release. Then, the samples in acetonitrile were centrifuged at 10 000 rpm for 10 min and supernatant (100 μL) gathered was diluted to 1 mL for encapsulation and loading detection as previously mentioned.
Fourier transform infrared spectroscopy (FTIR)
FTIR method was used to identify the functional groups related to structure and composition via absorption spectrum. The infrared spectra of the n-NUC, PLGA and NUC-PLGA-NPs, prepared as KBr discs, were measured over the range of 4000–400 cm−1.
Differential scanning calorimetry (DSC)
Thermograms of n-NUC, PLGA and NUC-PLGA-NPs were obtained with a differential scanning calorimeter (DSC 200 F3 Maia, Germany) and calibrated using a pure indium sample. Different samples (2–3 mg) were placed in aluminum pan heated up at 10 °C/min within 0–550 °C under dry nitrogen, respectively.
Size and zeta potential
The particle size and zeta potential of drug-free NPs and NUC-PLGA-NPs were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instrument, Worcestershire, UK). All samples were diluted in distilled water and equilibrated for 30 min before measurement. Each sample was analyzed in triplicate.
Transmission electron microscopy (TEM)
The morphology and size of the dried NUC-PLGA-NPs was measured using a TEM. A drop of NUC-PLGA-NPs suspension was placed onto a 200 mesh copper grid and dried at room temperature (RT) for view.
Release kinetics in vitro
A fixed weight of NUC-PLGA-NPs (100 mg) was dispersed in simulated gastric fluid (0.2% w/v NaCl in 0.7% v/v HCl at a pH of 1.2) and simulated intestinal fluid (pH 6.8) without enzymes, respectively. The solution was divided into 24 Eppendorf tubes with continuous shaking at 120 rpm at 37 °C. Aliquots (100 μL) were withdrawn and centrifuged at 10 000 rpm for 10 min after predetermined time intervals (1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h). After removing the supernatant, the pellet was resuspended with 1 mL methanol for HPLC detection.
Cellular uptake
For confocal laser scanning microscopy (CLSM, Leica Microsystems, Wetzlar, Germany) imaging, coumarin-6 (green fluorescence) was loaded as described in the section of preparation of NUC-PLGA-NPs, apart from the change of NUC to a hydrophobic fluorescent probe coumarin-6. HepG2 cells (1 × 104 cells/well) were seeded into confocal dish for 24 h. After incubated with free coumarin-6 or coumarin-6-loaded NPs for 4 h, cells were incubated with DIL (red fluorescence for general cell membrane labeling) for 20 min and then treated with Hoechst for staining of nucleus.
In vitro cytotoxicity assay
Cell proliferation of HepG2 treated with OA (0.04 mM as our previously used) and blank PLGA, n-NUC and NUC-PLGA-NPs supplemented with OA were measured using the CCK8 assay kit according to the manufacturer’s instructions. The concentration of NUC used in n-NUC and NUC-PLGA-NPs was 0.05 mM as previously used (Zhang et al., Citation2015). The untreated cells were selected as control. Briefly, 10 μL CCK8 solution was added to each well (100-μL medium) incubated with different formulations described above and incubated for additional 1.5 h at 37 °C. The absorbance was recorded at 450 nm with a microplate reader (Thermo Scientific, Rockford, IL).
Cell apoptosis analysis
Cells apoptosis of HepG2 treated with OA and PLGA, n-NUC and NUC-PLGA-NPs supplemented with OA were measured using the Annexin V-PE/7AAD Apoptosis Detection Kit following the manufacturer’s instructions. The untreated cells were selected as control. All samples were analyzed by flow cytometry (BD Accuri C6; BD Biosciences, San Jose, CA).
Oleic acid-induced hepatic steatosis
OA was selected to induce in vitro model of hepatic steatosis as previously described with slight modifications. Briefly, HepG2 cells were incubated with OA (0.04 mM) for 24 h to induce cellular steatosis. To determine the effect of NUC on OA-induced steatosis, cells were incubated with PLGA, n-NUC, NUC-PLGA-NPs and vitamin E (0.025 mM, selected as positive control) 24 h before treatment with 0.04 mM OA. The concentration of NUC used in n-NUC and NUC-PLGA-NPs was 0.05 mM and the untreated cells were selected as control. Cells were gently washed with phosphate buffered saline (PBS) three times and fixed using 4% paraformaldehyde at RT for 20 min. Subsequently, cells were washed with PBS three times, stained with freshly prepared working solution of ORO (diluted in double-distilled H2O at volume ratio of 3:2) for 30 min at RT, and redyed for 30 s in hematoxylin staining solution. Finally, cells were washed with PBS five times before examined under EVOS microscope (Thermo Fisher Scientific, Waltham, MA). The intracellular lipid accumulation was quantitated by Image-Pro Plus 6.0 (Media cybernetics Inc., Bethesda, MD).