Curcumin is a natural phenolic compound extracted from the herb
The liver is a vital organ which detoxifies various toxic substances, chemicals, and microbiological agents to which it is exposed. The morphological changes in the liver have a tendency to affect the metabolic events of the whole body, which is often associated with dysfunction of the detoxification process [
CCl4 is a well-known hepatotoxin that is widely used to induce toxic liver injury in a range of laboratory animals. CCl4-induced hepatotoxicity is believed to include two phases. The initial phase involves the metabolism of CCl4 by cytochrome P450 to the trichloromethyl radicals (CCl3⋅ and/or CCl3OO⋅), which leads to membrane lipid peroxidation and finally to cell necrosis [
Curcumin is a natural phenolic compound extracted from the herb
The nanoparticle is one of the novel drug carriers for therapeutic and diagnostic objectives which have several potential effects in improving accumulation and bioavailability of drug in target side, thereby suppressing immunogenicity and finally reducing adverse effects. Additionally, nanoparticles also promote drug solubility, controlled and sustained drug release, decreased drug elimination, and delivered more drug combination treatment for synergistic effect [
Nanoparticles such as liposomes, micelles, nanogels, and polymeric nanoparticles can be used to deliver therapeutic concentration of curcumin that enhances the therapeutic efficacy of curcumin. Grama et al. [
The aims of the current study were to synthesize and characterize Cur-NLs and to evaluate its protective role against CCl4-induced liver damage in mice. We employed thin film hydration method to prepare Cur-NLs with high entrapment efficiency and drug loading efficiency. Experiment in vivo demonstrated that Cur-NLs display a better hepatic protective effect on CCl4-induced acute liver injury in mice compared with free curcumin administration. Cur-NLs exhibiting better hepatic protective effect might be due to the slow and regular release of curcumin by nanoparticles.
Curcumin was purchased from Xi’an Tianbao Biotechnology Co. Ltd. Soybean phosphatidylcholine (SPC) and cholesterol (CHOL) were purchased from AVT Pharmaceutical Technology Co. Ltd. Dichloromethane, methanol, and CCl4 were purchased from Tianjin Fenghua Chemical Reagent Technology Co. Ltd. Alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) measurement kits were purchased from Nanjing Jiancheng Bioengineering Institute.
Male Balb/c mice (SPF) were purchased from the Laboratory Animal Center in the Academy of Military Medical Sciences (Beijing, China) at 6 to 8 weeks old with an average weight of 18~22 g. The animals were housed in a standard environmental condition and were provided ad libitum access to food and water. All procedures with animals were in strict accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH publications no. 8023, revised 1978) and approved by the Animal Care and Use Committee of Yanshan University, China (Ethics number: YD2017011).
Cur-NLs were prepared according to an established method [
A TEM (JEM-100CX/II) was used to observe the size and the morphology of the Cur-NLs. The samples for TEM were prepared by a standard procedure. 2% of ammonium molybdate was used as a staining agent. Then the carbon film-coated copper grid was placed on the samples for 10 min, and the excess solution was removed with a filter paper. The samples were air-dried and then observed under the TEM.
Malvern Zetasizer ZS (Malvern Instruments, UK) was used to measure the sizes and surface zeta potentials of the Cur-NLs. The mean liposome diameters and zeta potentials were determined by dynamic light scattering (DLS) and electrophoretic mobility measurement, respectively. All characterization measurements were repeated three times at 25°C.
X-ray diffraction was performed using X-ray diffractometer (Model PW 1710) control unit Philips Anode material Cu, 40 kV, 30 MA, which Cu K
Potassium bromide (KBr) technique was used for FTIR analysis. First, KBr was dried at 105°C and grounded finely. The curcumin, physical mixture of curcumin excipient, and Cur-NL samples were added to it (sample: KBr = 1 : 3), respectively, and were finely grounded again. They were compressed under high pressure to prepare pellets of 10.0 mm and 1-2 mm thick. The pellets were scanned over a range of 4000 cm−1 to 400 cm−1 and spectra were recorded using FTIR (Jasco FTIR 460 Plus, Japan) [
Firstly, the regression curve of concentration vs absorbance in 425 nm was obtained to analyze the concentration of resulting curcumin solution after ultrafiltration. Then Cur-NLs were put in ultrafiltration centrifuge tube (Millipore, 10 KD) and centrifuged to separate the unentrapped curcumin from Cur-NLs (5000 rpm, 15 min). Subsequently, 1 mL of resulting free curcumin solution was transferred into a new centrifuge tube. The curcumin concentration was determined as described above after 2 mL of methanol was added into this tube. EE and DL were calculated with the following formulas [
Male Balb/c mice were randomly divided into five experimental groups with 8 mice in each, including normal control group (PBS, 0.2 mL), CCl4 model control group (PBS, 0.2 mL), free curcumin treatment group (free-Cur, 2 mg/kg), curcumin nanoliposome treatment group (Cur-NLs, 20 mg/kg ≈ 2 mg/kg pure curcumin), and positive control (silybin, 50 mg/kg). All the grouped animals were administrated with above different drugs or PBS by tail vein injection for 14 days. Except the normal group, mice in the other 4 groups received single dose of 2% CCl4 in peanut oil (v/v, 0.3 mL) intraperitoneal injection on the 14th day. Normal control received equal amount of vehicle instead of CCl4.
On the 15th day, the mice were anesthetized and sacrificed. The liver, kidney, thymus, and spleen tissues were immediately removed and weighed. The organ index was calculated according to the following formula.
To test liver function, we examined the serum levels of ALT, AST, and ALP. Before execution, the blood of the experimental mice of control and treated groups was collected in Eppendorf tubes, deposited for 1 h at 4°C, and centrifuged for 15 min at 3000 rpm, and then the serum was collected. ALT, AST, and ALP activities were assayed according to the instructions of the kits.
The liver tissues of mice were fixed by 10% formaldehyde and embedded in paraffin. The slices were cut into 4
After execution, the livers of the experimental mice in all groups were removed. The liver homogenates (10.0%, w/v) were prepared in the normal saline. The resulting suspensions were centrifuged at 4000 rpm for 15 min, and the supernatants were collected for further measurement. All treatments were conducted in an ice bath or at 4°C. The SOD, GPx, and CAT were then determined by commercial reagent kits according to the instructions. Lipid peroxidation in liver tissues was measured by the formation of MDA and it was estimated by commercial reagent kit as well.
All experiments were performed in three groups of parallel experiments and analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Multiple comparisons were performed using variance analysis and considered
The morphology of Cur-NLs was observed by TEM. Figure
Characterization of curcumin-loaded nanoliposomes. (a) TEM images of curcumin-loaded nanoliposomes. (b) Size distribution of curcumin-loaded nanoliposomes assessed by dynamic light scattering (DLS). (c) XRD of curcumin, physical mixture of curcumin and excipients, and curcumin-loaded nanoliposomes. (A) Curcumin. (B) Physical mixture of curcumin and excipients. (C) Curcumin-loaded nanoliposomes.
The particle size of Cur-NLs was measured by laser particle sizer, and the average size of Cur-NLs was
When X-rays are diffracted by crystals, each crystalline material has its own unique diffraction pattern, and their characteristics can be characterized by the relative intensity of the diffraction I/I0.
The diffraction peaks at different angles of the curcumin monomer crystals can be observed in the X-ray diffraction pattern and the peaks of the physical mixture of curcumin and excipients were reduced, but these diffraction peaks disappeared in the Cur-NLs. This result proved that curcumin had been highly dispersed in liposomes (Figure
In order to determine the interaction between the drug and the excipients, the compatibility of the drug and the excipients was further determined by infrared spectroscopy. The infrared spectra of curcumin, physical mixture of curcumin and excipients, and Cur-NLs are shown in Figures
FTIR spectrum of curcumin, physical mixture of curcumin and excipients, and curcumin-loaded nanoliposomes. (a) FTIR spectra of curcumin. (b) FTIR spectra of physical mixture of curcumin and excipient. (c) FTIR spectra of curcumin-loaded nanoliposomes.
The infrared spectra showed that characteristic peak of curcumin appears at 3420 cm−1 for phenolic hydroxyl groups, 1630 cm−1 for C = O symmetrically stretch, 1510 cm−1 for the C = O and C = C stretch, 1280 cm−1 for -C-O of the aromatic ring stretch. Compared with the characteristic peaks of curcumin, the characteristic peaks of physical mixture of curcumin and excipients and Cur-NLs both showed 1510 cm−1 and 1280 cm−1 red shift to 1470 cm−1 and 1240 cm−1. This suggested that the olefin bond and the aromatic ring of curcumin have a chemical bond with the phospholipid [
The Cur-NLs were prepared by ultrasound membrane dispersion method. After ultrafiltration, the solution was determined at 425 nm using spectrophotometer. The EE of Cur-NLs was
Livers and kidneys are important organs of body metabolism. The results in Figure
Effects of curcumin-loaded nanoliposomes on organ index and biochemical parameters of liver tissues. (a) Effect of curcumin-loaded nanoliposomes on organ index in mice. (b) Effect of curcumin-loaded nanoliposomes on serum ALT, AST, and ALP activities in mice.
Hepatoprotective effects of Cur-NLs were determined by quantitative analysis of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) levels as shown in Figure
After 14 days of treatment, liver tissues of all groups were taken from the animals of all groups and were subjected to histological analysis (Figure
Photomicrographs of liver sections stained by HE from (a) normal control mice showing that the hepatocytes are polyhedral in shape, with central rounded vesicular nuclei and a acidophilic granular cytoplasm (×100); (b) CCl4 model control mice showing disorganized hepatic architecture with multiple areas of necrosis with apoptotic cell and apoptotic body distinguished by dense eosinophilic cytoplasm and pyknotic nucleus and mononuclear cellular infiltration around the portal tract with branching of bile ductules and proliferation (×100); (c) free curcumin treatment mice showing mild mononuclear cellular infiltrate mildly dilated and congested portal vein and normal bile duct (×100); (d) curcumin-loaded nanoliposome treatment mice showing organized hepatic architecture with vesiculated nuclei and preserved central vein and a few numbers of hepatocytes show deeply stained acidophilic cytoplasm and dark nuclei (×100); (e) silybin treatment mice showing more or less normal portal vein, preserved bile ducts, and a few numbers of hepatocytes show deeply stained acidophilic cytoplasm and dark nuclei (×100).
In order to explore the protection mechanisms behind Cur-NLs on the liver toxicity induced by CCl4 in vivo, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels in the mice liver tissues were further determined.
The present data shown in Figure
Effect of curcumin-loaded nanoliposomes on SOD, GPx, and CAT activities and MDA level in the mice liver tissues.
Pharmaceutical nanocarriers such as liposomes have demonstrated enhanced in vivo stability, longer circulation times, better permeability, resistance to metabolic processes, and high efficiency of drugs. The benefits of nanoscale delivery systems are supported by numerous preclinical and clinical data [
In the current study, Cur-NLs were synthesized to enhance the water solubility of curcumin and to decrease the particle size which result in the formation of a thinner hydrodynamic layer around particles and increase the surface specific dissolution rate. The DLS analysis of the aqueous dispersion of the prepared Cur-NLs revealed the formation of nanoparticles with an average diameter of
CCl4 is an effective hepatotoxic agent, and even a single exposure can promote severe liver toxicity, including necrosis and steatosis. Hence, CCl4 is widely used as a model for evaluating the hepatoprotective activity of new drugs or drug formulations [
It is well known that AST, ALT, and ALP are sensitive indicators of liver injury. After the administration of CCl4, reductive dehalogenation of CCl4 catalyzed by cytochrome P450 will form a highly reactive trichloromethyl free radical (CCl3) and then form the trichloromethyl peroxy radical (CCl3OO) as the precursor of lipid peroxidation [
There are several intrinsic antioxidative defenses of the cells, and one of which is the existence of antioxidant enzymes such as SOD, CAT, and GPx. In the present study, intraperitoneal injection of CCl4 obviously downregulated the expressions of these enzymes and induced oxidative stress as one of the by-products of its metabolism. However, these enzymes were restored to their basal levels after the pretreatment with free-Cur, Cur-NLs, or silybin followed by CCl4. Because of its polyphenolic structure and a
We proved here that Cur-NLs successfully ameliorated CCl4-induced hepatotoxicity in mice as demonstrated by decreased levels of the hepatic injury markers ALT, AST, and ALP. The observed protective effects of Cur-NLs may be due to antioxidant effects as shown by the reduced lipid peroxidation (MDA level), increased SOD, CAT, and GPx activities. The histopathological analysis conducted in this study also revealed dramatically protective effects on liver damage induced by CCl4.
Cur-NLs were found to be more effective in curing liver damage than free-Cur. This might be due to the fact that lipid bilayers of liposomes improved the drug permissiveness into the cell membrane and enhanced the penetration of Cur-NLs and the slow and regular release of Cur by nanoparticles that provide Cur with a rise in bioavailability, which, in return, increases therapeutic effects.
Compared with the previous study about curcumin-loaded nanoliposomes, the drug loading, stability, and entrapment efficiency of Cur-NLs prepared in this study were enhanced markedly. Furthermore, we discussed the protective effect and possible mechanism of Cur-NLs on liver systematically. These observations imply that Cur-NLs act as a promising hepatoprotective agent in reducing liver oxidative stress produced by different stress factors.
Tetrachloromethane
D-Galactosamine
Curcumin-loaded nanoliposomes
Alanine transaminase
Aspartate transaminase
Alkaline phosphatase
Superoxide dismutase
Catalase
Glutathione peroxidase
Malondialdehyde
Entrapment efficiency
Drug loading
Polydispersity index.
No data were used to support this study.
Jian Li and Kun Li’s present address is Department of Biological Engineering, College of Environmental and Chemical Engineering, Yanshan University, 438 Hebei Street, Qinhuangdao City, Hebei Province 066004, China.
The authors declare that they have no direct financial relation with the commercial identities mentioned in this paper that might lead to a conflict of interests for any of the authors.
This work was financially supported by the Qinhuangdao Science and Technology Research and Development Plan (nos. 201101A132, 201705B024, 201701B044, and 201801B035) and by the Hebei Science and Technology Research and Development Support Program (no. 17272402D).