Pharmacokinetic
For the pharmacokinetic study, 18 male SD rats weighing 200–240 g were randomly assigned to three groups (n = 6) for IV administration of 0.2 mg/kg n-NUC, oral administration of n-NUC or NUC-PLGA-NPs at NUC concentration of 5 mg/kg, respectively. Blood samples (∼200 μL) were collected into heparinized tubes at 2 min, 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h after IV administration and at 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 6 h, 9 h and 12 h after oral administration. The blood samples were immediately centrifuged at 8000 rpm for 5 min. The plasma was separated by centrifugation and stored at −80 °C before analysis by HPLC-MS/MS, of which parameters were set as previously used (GU et al., Citation2014).
The peak concentration (Cmax) and the time of peak concentration (tmax) were observed from experimental results, and pharmacokinetic parameters such as half-life time (t1/2), total area under curve (AUCt) were processed by non-compartmental analysis using WinNonlin software (Certara, Princeton, NJ, USA). The absolute bioavailability (Fabs) was calculated as follows: Fabs (%) = (AUCig ×Doseiv)/(AUCiv ×Doseig) × 100%, while the relative bioavailability (Frel) was calculated by the following formula: Frel = (AUCA ×DoseB)/(AUCB ×DoseA) × 100%.
The effect of NUC on male Sprague Dawley rats with high fat diet-induced hyperlipidemia
After adaptive feeding for 7 days, rats were randomly distributed into four groups.
Group I: Normal control (NC) group fed a regular laboratory diet.
Group II: Hyperlipidemia control (HC) fed high-fat diet (HFD, consisting of 68.5% standard laboratory chow, 15% carbohydrate, 10% lard, 5% yolk powder, 1% cholesterol, and 0.5% sodium cholate.
Group III: Rats fed HFD supplemented with n-NUC (10 mg/kg/day).
Group IV: Rats fed HFD supplemented with NUC-PLGA-NPs at NUC of 10 mg/kg/day.
After 2, 4, 6 and 8 weeks of treatment, blood was collected by retro-orbital puncture and plasma was separated by centrifuged at 1000 rpm for 15 min. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were assayed by using kits purchased from Bio-Swamp (Shanghai, China).
Statistical analysis
The data were expressed as the mean ± SD. Statistical analysis was performed using Student’s t-test and one-way ANOVA. A value for *p < 0.05 was considered statistically significant.
Results and discussion
Preparation and characterization of NPs
Oral administration is widely used due to its low-associated costs and high-patient compliance as compared to other routes like IV, intramuscular and subcutaneous injection. However, oral drugs with poor aqueous solubility and sensitive to degradation are unable to reach the minimum effective concentration exhibiting therapeutic effect. Nanotechnology-based drug delivery system has shown to improve solubility and shield against digestive enzymes and pH changes, among which PLGA is particularly interesting for oral drug delivery due to its biocompatibility, degradability in physiological environments, commercial availability of various grades and the safe biodegradation products. NUC, a raw material in Chinese medicinal herb, which is an aporphine alkaloid extracted from lotus leaves contributing to lipid metabolism. In this study, NUC powder was suspended in the PLGA solvent composited of dichloromethane and acetone at a volume ratio of 3:2. Basing on the emulsion encapsulation techniques, this suspension was emulsified in an aqueous solution containing BSA. Organic solvent was then evaporated, after which nanoscaled products were finally washed and lyophilized.
Encapsulation efficiency and drug loading
The EE% and DL% of NUC-PLGA-NPs at various NUC feeding were shown in . With increasing concentrations of NUC, the EE% decreased, while the DL% increased first and then decreased sharply. The decrease of EE% might be attributed to excessive drug, which hampered the overall stability of the formed NUC-PLGA-NPs after PLGA reached the saturation solubility in the polymer matrix (Xin et al., Citation2010). Fortunately, the DL% could reach 8.89 ± 0.71% along with EE% at 88.54 ± 7.08% when adding 8 mg NUC. Based on these results, we optimized the feeding amount of NUC at 8 mg when 100 mg PLGA was used in the following experiments.
Figure 1. (A) Encapsulation efficiency (EE%) and drug loading (DL%) of nanoparticles based on various amount of NUC adding (mean ± SD, n = 3). FTIR spectra (B) and DSC curves (C) of NUC, PLGA and NUC-PLGA-NPs. Size distribution (D) and zeta potential (E) of PLGA. Size distribution and a representative TEM image (F) and zeta potential (G) of NUC-PLGA-NPs. (H) In vitro release of NUC from NUC-PLGA-NPs in simulating gastric fluid at pH 1.2 and in simulating intestinal fluid at pH 6.8 over a period of one week (mean ± SD, n = 3).
FTIR spectroscopy
FTIR spectra of n-NUC, PLGA and NUC-PLGA-NPs were shown in . The spectrum of PLGA showed a characteristic peak at 1757 cm−1 which indicated unconjugated carbonyl (C=O) stretching (Zabelin et al., Citation2016). The NUC showed peaks in the range of 1450–1600 cm−1, which belonged to frame vibration of benzene ring. NUC-loaded NPs showed peak at 1680 cm−1 corresponding to PLGA and characteristic peak of benzene ring with moderate peak shifting, which confirmed the encapsulation of NUC in PLGA.
DSC
DSC thermograms of n-NUC, PLGA and NUC-PLGA-NPs are shown in . The drug showed a sharp peak at 165 °C consistent with its melting point of crystalline regions, while PLGA exhibited an endothermic relaxation peak at 50 °C corresponding to its glass transition temperature (Gidwani & Vyas, Citation2016). Owing to the fact that PLGA is amorphous in nature, there was no distinct melting point. NUC-PLGA-NPs exhibiting the similar relaxation peak as PLGA and the characteristic peak of n-NUC were not viewed in NUC-PLGA-NPs, indicating that drug was encapsulated by PLGA in an amorphous or disordered-crystalline phase of a molecular dispersion or a solid solution state in the polymer matrix.
Size and zeta potential
After re-dispersion in deionized water, drug-free NPs and NUC-PLGA-NPs were characterized concerning average size, width of distribution and zeta potential. A typical DLS size and zeta profile on the distribution of NPs and NUC-PLGA-NPs were shown in , respectively. The average size of empty NPs was 130.58 ± 5.36 with polydispersity index (PDI) of 0.26 ± 0.028, while an increase size of 150.83 ± 5.72 with PDI of 0.24 ± 0.025 was observed for NUC-PLGA-NPs, probably for the presence of NUC entrapped into the core of PLGA. Nanoparticles of this size not only can be internalized by endocytosis but also are large enough to be maintained in circulation for a long period (Anselmo & Mitragotri, Citation2016). More negative (−) or positive (+) electricity prevented the particles from aggregation due to the repelling interaction (Honary & Zahir, Citation2013; Gossmann et al., Citation2015). Zeta potential values of drug-loaded NPs and unloaded ones were −23.45 ± 1.54 and −22.73 ± 1.63 mV, respectively, indicating the stability of NUC-PLGA-NPs in suspension. NUC-PLGA-NPs exhibit spherical morphology with low tendency of agglomeration. The TEM-observed size was around 100 nm smaller than DLS-calculated size, because the latter size refer to the hydrodynamic diameter of the nanoparticles with a polymer layer, while the former one represented only the core of the nanoparticles (Han et al., Citation2016).
Release kinetics in vitro
In vitro release of NUC from NUC-PLGA-NPs was determined by imitating digestion conditions in gastric and intestinal juice, respectively. As shown in , NUC release fast within the first 12 h both in simulating gastric fluids and intestinal fluids, may be due to the presence of loosely bound NUC on or near the surface of particles (Sun et al., Citation2015), followed by a very slow release up to 45.95 ± 5.2% in artificial gastric fluids and sustained and prolonged release up to 77 ± 6.67% in artificial intestinal fluids. Acidic environment is responsible for the protonation of the carboxyl groups of PLGA and the aggregation of the nanoparticles, which shapes into a stable structure to limit NUC release from the nanocomposites. While the increased pH in artificial intestinal juice triggered NUC release from the NUC-PLGA-NPs due to the increased water absorption of GA group, which induced the penetration of the water toward the core of nanocomposites. On the basis of the results above, we concluded that minimal amounts of NUC could be released from the nanocomposites in the stomach when given orally, while the residues could achieve a sustained release and effective treatment.
Cellular uptake
To illustrate the intracellular delivery of NUC by PLGA, coumarin-6 (green fluorescence) was encapsulated into NPs on account of the hydrophobic property as NUC. In , CLSM images show cell membranes stained in red with DIL and general nucleus labeled by Hoechst (blue fluorescence). Free coumarin-6 could hardly be taken up by HepG2 cells, while cells showed significantly stronger fluorescence signal after incubated with coumarin-6-loaded NPs. These results indicate the excellent role of NUC-PLGA-NPs in the treatment of alleviating lipogenesis, for their perinuclear location might be important for higher drug concentration to address excess lipid accumulation within hepatocytes.
Figure 2. Cellular uptake of free coumarin-6 (A) and coumarin-6-loaded NPs (B) against HepG2. Scale bar: 50 μm.
Cytotoxicity and apoptosis of nanoparticles
Prior to evaluating the reduction of the n-NUC and NUC-PLGA-NPs on lipid droplet accumulated in OA-induced hepatic steatosis, their impact on cell viability and apoptosis against HepG2 was tested in order to avoid any misinterpretation due to cytotoxicity. HepG2 cells incubated with n-NUC and NUC-PLGA-NPs supplemented with OA showed cell viability above 90% (). Concentration of OA at 0.04 mM and NUC at 0.05 mM reveals no significant difference compared with the control group. For better comparison among different drugs, OA at 0.04 mM and NUC at 0.05 mM both in n-NUC and NUC-PLGA-NPs were used for all the subsequent experiments.
Figure 3. Cell viability (A) (mean ± SD, n = 3) and apoptosis (B) of HepG2 cells treated various formulations. (C) The effect of NUC on TG accumulated HepG2 of OA-induced hepatic steatosis. (D) Relative integrated option density (IOD) value was quantitated using Image Pro Plus 6.0 (mean ± SD, n = 3). *p < 0.05.
Annexin V-PE/7AAD is used to quantitatively determine the percentage of cells undergoing apoptosis. As shown in , few apoptotic cells were detected in the control group and no significant increase in apoptotic cells was observed in the cells treated with various formulations, suggesting that it is possible to model a liver steatosis with distinct lipid accumulation and without apparent cytotoxicity and apoptosis.
NUC reduced TG accumulation in steatotic HepG2 cells
To examine the reduction of NUC on TG accumulation in OA-treated HepG2 cells, steatotic HepG2 cells were incubated with various treatments and evaluated using ORO staining. The steatosis was simulated in vitro by treating HepG2 cells with OA. Both OA, a monosaturated omega-9 fatty acid, and palmitic acid (PA), a saturated fatty acid, are the dominating dietary free fatty acids. Owing to the more steatogenic but less apoptotic nature of OA to hepatic cells than PA, OA was widely used to induce liver steatosis (Cui et al., Citation2016). As shown in , intracellular lipid droplets were positively stained with ORO solution of OA-treated cells and negatively stained in cells without OA treatment. The low level of OA at 0.04 mM is able to cause prominent ORO-stained lipid droplets in the cytoplasm of cells, which might be due to the deficiency of communication between HepG2 and other types of cells, particularly adipocytes. As previously demonstrated, OA treatment exhibited a dose-dependent increase in lipid accumulation and cytotoxicity. Considering both the lipid accumulation and cytotoxicity, 0.04 mM was selected as the optimal concentration for the induction of lipid accumulation in HepG2 cells as a model of hepatic steatosis. As shown in , TG was markedly accumulated in OA-treated cells compared with NCs. n-NUC and NUC-PLGA-NPs could decrease the intracellular TG content when compared to OA group (*p < 0.05). In addition, NUC-PLGA-NPs are more efficient against OA-induced TG accumulation, which was comparable with that of vitamin E and the percentage of inhibition is significantly higher than n-NUC (*p < 0.05) due to improved water solubility and sustained release. The first stage of non-alcoholic fatty liver disease (NAFLD) has nothing to do with excess alcohol consumption, but closely associated with the accumulation of lipids in hepatocytes. TG deposition, the primary pathological feature of liver steatosis, is regarded as the first stage in the evolution of NAFLD. In our previous study, we have demonstrated that NUC contributed to the inhibition of TG accumulation, while the better coordinating role of NUC-PLGA-NPs on TG over-accumulation has been demonstrated in this study.