Hepatocellular carcinoma (HCC) is the sixth highest incidence cancer worldwide, accounting for 5.7% of the overall incident cases of cancer with the third leading cause of cancer-related deaths in all age group population1. The incidence of HCC varied widely due to the geographic variations, many of these cases occur in developing countries (82%, with 366,000 new cases estimated in males and 147,000 in women, in 2002) as compared with the developed countries (74,000 new cases in men and 36,000 in women)2,3. In fact, liver cancer is the third most common cancer in the developing countries among men, after lung and stomach cancer. Also, it is between 2 and 8 times more common in men than in women. China alone accounts for 55% of the new cases of liver cancer, other high incidence areas being sub-Saharan Africa, Japan, and South-East Asia. Between the years 1978 and 1992, while some centers in high-risk countries like China, India, and Spain recorded a decreasing incidence of liver cancer by as much as 30%, other centers in predominantly low-risk populations like Italy, Australia, and France recorded a nearly 100% rise in the number of cases of primary liver cancer4. Active targeting nanoparticle delivery systems have become a potential new drug modality with the opportunity of enhanced water solubility, constant and stable release of the drug, tumor-specific accumulation, improved antitumor efficacy, and reduced nonspecific toxicity5. Metastasis is still the primary cause of severe morbidity and mortality in cancer. Matrix metalloproteinases (MMPs) play an important role in cancer progression, including tumor growth, invasion, metastasis, and angiogenesis. For this reason, treatment methods aiming at MMPs have great significance6. Gelatinases may be suitable candidates for stimuli-responsive targeted strategies. During development of carcinogenesis, tumor cells participate in several interactions with the tumor microenvironment involving extracellular matrix, growth factors and cytokines associated with extracellular matrix, as well as surrounding cells (endothelial cells, fibroblasts, macrophages, mast cells, neutrophils, pericytes, and adipocytes).7 Four hallmarks of cancer that include migration, invasion, metastasis, and angiogenesis are dependent on the surrounding microenvironment. The expression of MMPs in the tumor microenvironment depends not only on the cancer cells, but also on the neighboring stromal cells5,8. MMPs exert their proteolytic activity and degrade the physical barriers, facilitating angiogenesis, tumor cells invasion, and metastasis. Tumor growth and angiogenesis also depend on the increased availability of signaling molecules, such as growth factors and cytokines, by MMPs making these factors more accessible to the cancer cells and the tumor microenvironment8. The principle aim of this study is to evaluate the antimetastatic effect of pemetrexed-loaded poly(ethylene glycol) (PEG)-Peptide-poly(E-caprolactone) (PCL) nanoparticles in experimental HCC model in vitro.
Material and methods
Pemetrexed was the generous gift from M/s Eli Lilly and Company. HepG2 was acquired from the NCCS Pune and IGIB Delhi. Di-ethylnitrosamine (DEN)-CCl4 used for mode development was bought from M/s Sigma PVT. Ltd (MDL number MFCD00013890). All other chemicals and reagents used in the present study were of analytical grade. Distilled water was used in the study.
Preparation of PEG-PCL nanoparticles
Drug loaded nanoparticle formulation was prepared by solvent evaporation method as described by Lu et al9 briefly predetermined amounts of mPEG-N-hydroxyl sulfo succinimide sodium salt and peptide were dissolved in dimethylformamide containing 3% triethanolamine and the mixture was stirred for 3 hours at room temperature. The obtained solution was filtered by using filter membrane (3500 molecular weight cutoff; Sigma) for 24 hours to remove the nonreacted peptide. Amination of PCL-COOH was done in polymerization tube at 160°C for 24 hours. The prepared solution was then precipitated with ethanol and dried in vacuum. PEG-Peptide-PCL nanoparticles and PEG-PCL nanoparticles were then prepared by adding predetermined amount of PCL-NH2, PEG-Peptide, with the 1-ethyl-3-(3 dimethylaminopropy1) carbodiimide hydrochloride and N-hydroxyl sulfo succinimide sodium salt.
Preparation of pemetrexed-loaded nanoparticles
A total of 5 mL of vehicle and pemetrexed were dissolved in 1 mL of dichloromethane. The mixture was emulsified in 3 mL of 5% PVA solution (w/v) by sonication for 30 seconds and was converted into an o/w emulsion. The obtained emulsion was added into 8 mL of 1% PVA solution (w/v) and handled by sonication again, then stirred to remove dichloromethane at room temperature in a fume cupboard. The resulting solution was filtered through filter membrane to remove nonincorporated drugs and copolymer aggregates. The nanoparticles solution was freeze dried with 3% mannitol and saved at 4°C. Drug-free nanoparticles were produced in a similar way without adding pemetrexed.
Healthy swiss albino mice of 10–15 days were procured from the animal house of DIPSAR. They were housed in polypropylene animal cages and acclimatized for 3 days with 12 hours day-and-night cycle before the model development at a relative humidity of 50%–55% and temperature of 25–27°C. Standard rodent diet was given to the subjects. Drinking water was provided ad libitum. All the animal protocols were duly approved by the DIPSAR Animal Ethics Committee protocol no. IAEC/2015-I/protocol no. 13 and were performed with care.
All data were given as the mean±SD. Experimental results were analyzed by Student t test and 1-way analysis of variance with Student-Newman-Keuls method (Software used: GraphPad Prism Version 6). The survival of mice was demonstrated using the Kaplan-Meier method and the log-rank statistical test. P<0.05 was considered as the level of significance for values obtained for treated compound to control.
Results and discussion
The nanoparticle formulation of pemetrexed-loaded PEG-Peptide-PCL nanoparticles, possessed the mean vesicular size of 385±7.3 nm (Table 1), encapsulation efficiency of 84.21% followed by 22.85% of drug loading content (Table 2), and zeta potential of −25.5 mV. The release study done for the formulation has shown that the rate and amount of pemetrexed released from the pemetrexed-loaded PEG-PCL-NP were strongly dependent on the pH of the medium. Pemetrexed-loaded PEG-PCL-NP showed much faster drug release at pH 4.5 and 5.5 than at pH 7.4. At the end of 24 hours, 81.2%±3.2%, 53.2.82%±2.3%, and 38.2%±2.5% of pemetrexed was released in acetate and phosphate buffer, respectively (Graph 1A), the release of drug from the gelatinase NP was better in comparison with only binder (Graph 1B).
Attenuated total reflectance- Fourier-transform infrared spectroscopy was done for characterization of nanoparticles where solid/liquid form of samples were used, which has shown significant absorption of polymer and the antibody followed by the quick absorbance of drug. A conjugate there appeared a single intermediate strong peak centered at 1650 cm−1 (amide band). These changes may indicate that N-H moieties of the protein are directly involved in its interaction with the drug surface. Also, the regions of C-NH2 vibrations are enhanced near 1400 cm−1. Peak at 1765.24 and 1663.84 cm−1 is for the pemetrexed and PCl (Fig. 1A). However, a weak broad feature at 2520 cm−1 (typically of the -S-H stretching vibrations of amino acid residues) was also observed in the conjugate, showing that not all the amino acids were bound to the surface. Amino acids stretching are seen in 1700–1500 cm−1 (Fig. 1B).
Zymography was performed to measure the MMPs activities in carcinoma cells. The results of zymography and indicated that the carcinoma cells secreted MMP-9 proteins.
For the study 10–15 days old mice was procured from the animal house of DIPSAR at day one 90 mg/kg of DEN injection intraperitoneally was given. And since then they were fed with the CCl4 water (1:5). Cells were cultivated in Dulbecco’s Modified Eagle’s Median supplemented with 10% fetal bovine serum and maintained under a 5% CO2 humidified atmosphere at 37°C. Cells growing exponentially in vitro were trypsinized and harvested for tumor implantation. All animal manipulations were performed under sterile conditions.
To create orthotopic HCC models, mice were anesthetized with intraperitoneal injection of a ketamine/diazepam solution (50 mg/kg ketamine and 5 mg/kg diazepam) and operated on a prewarmed operation table. A subcutaneous dose of Baytril 5 mg/kg injection was given before operative procedure. Briefly, an upper midline laparotomy was performed.
The portal vein was exposed by displacing the duodenum through a midline incision of the abdomen and a suspension of 1×106 HepG2 cells in Dulbecco’s Modified Eagle’s Median medium was injected via the portal vein using a 30-G needle within a period of a minute. The abdominal incision was sutured close with 5/0 maxon (monofilament polyglyconate synthetic absorbable suture) and skin was closed with 5/0 prolene (polypropylene suture). Postoperatively, the mice were given a subcutaneous injection of 5 mg/kg Caprofen for analgesia. Alpha fetoprotein (AFP) test was performed as the tumor biomarker.
AFP test was done to confirm the presence of HCC in mice models. Blood was withdrawn from the retro-orbital route on the second day of injection of cell line. The level of AFP found in normal group was in range of 9–12 ng/mL, where the case of control and other treatment groups is the range of AFP was between 213 and 238 ng/mL. After the treatment the level of AFP was decreased in almost all the groups in the range of 98–112 ng/mL (Table 3).
The body weight of mice recorded during different intervals of the study protocols is shown in (Table 3). Significant reduction in the body weight of mice was observed in DEN/HepG2 groups before treatment. However, after commencing the treatment the changes in body weight variations indicated the acceptable security of the pemetrexed-loaded nanoparticles.
Blood was withdrawn on day 20 by retro-orbital plexus method form all groups. Different hematological parameters analyzed are as, hemoglobin, white blood cell count, red blood cell count, by standard procedures (Table 3 and Fig. 2). To reduce the hematological side effects 0.8 mg/kg folic acid were given orally on alternate days to all the treatment groups except disease control group and normal group.
Survival rate of the mice were assessed by calculating it by Kaplan-Meier estimation and log rank test (P<0.001) decreased significantly in controlled group as compared with the normal. Treatment with HDNP lead to higher survival rate then the free pemetrexed treated group (Graph 2).
In normal mice (Fig. 3A) light microscopy showed a normal parenchyma. Hepatic laminae spread out regularly from central veins. Connective tissue lining of hepatic lobules were not observed. Network sinusoids appeared continuous and interconnected. Necrosis and fibrosis were absent.
In DEN-CCl4-treated mice (Fig. 3B), the liver appeared diffusely nodular. Fibrosis was present in uniform septa of connective tissue and thin fibrous capsules defined the nodules. Necrosis areas were present. In pemetrexed treated group (Fig. 3C) the hepatic laminae started out; however, the necrosis was bit present. In low and high dose NP treated (Fig. 3D & E, respectively), livers showed recovery to normal parenchymal architecture with the disappearance of nodular shapes. Connective tissue was organized into fibrotic bundles. Necrosis was almost absent. NP treated groups has shown lesser amount of vasculogenesis in intestine and stomach region (Fig. 3F). Figures 3G and H shows the formation of ascites fluid in peritoneal cavity followed by the vasculogenesis in stomach and intestine area.
To observe the role played by NP in modulating the carcinoma microenvironment with respect to the soluble mediators of immune system; we quantified the cytokine levels in the tissue homogenate. The levels of VEGF, IL-12, and TNF-a were shown in Figure 4. The levels of VEGF and TNF-a were decreased (P<0.001) in a dose-dependent manner as compared with control group (Figs. 4A, B). NP treatment increased the IL-12 levels (P<0.05) (Fig. 4A), when administered at a dose of 25 mg/kg.
Liver toxicity is a common side effect of pemetrexed. However, which could be managed easily while providing pemetrexed regimen9. During pemetrexed treatment in first-line setting ∼70% patients develop liver toxicity, which requires delay in treatment or dose reduction from the subsequent cycle9. The targeted delivery of pemetrexed reduces the side-effects and increases the survival time. AFP A biomarker, an oncofetal serum protein, is progressively lost during development, such that it is virtually absent from the healthy mice. It has long been recognized that exposure of rats to certain carcinogens like DEN causes an elevation of circulating AFP levels10,11. AFP is a protein of fetal component produced during the embryonic period by the visceral endoderm of the gestational sac and, later on, by the liver. Its reexpression in patients with HCC has been described for over 40 years. Some studies have demonstrated that the presence of elevated levels of AFP in patients with liquid chromatography is a risk factor for the development of HCC10,12. Thus suggesting that increased AFP-production in patients with liquid chromatography might reflect, largely and abnormal or altered liver cell regeneration. High AFP serum levels have been found in 60%–70% of patients with HCC. Biochemical marker enzymes are used to screen particularly cancer conditions for differential diagnosis, prognosis, monitoring the progress and for assessing the response to therapy. These enzymes are more unique and changes in their activities reflect the effect of proliferation of cells with growth potential and its metabolic turnover9. In cancer conditions, there will be a disturbance in the transport function carried out by cell organelles including hepatocytes, resulting in the leakage of enzymes due to altered permeability of plasma membrane, and thereby causing a decreased level of these marker enzymes in the cells and increased level in serum. The structural integrity of the cells has been reported to be damaged in toxicity induced animals and this results in cytoplasmic leakage of enzyme into the blood stream.13 In this study, increase in pathophysiological marker enzyme levels upon DEN induction might due to disturbance in the transport function and the leakage of the enzyme. NP administration rectified the disturbance and enzyme leakage. SOD is said to act as the first-line of defense against superoxide radical generated as a by-product of oxidative phosphorylation. Cytotoxicity tests showed no significant differences in cytotoxicity among these therapeutic groups.
The signs of stimulation of apoptosis were noted by the presence of liver cell with shrunken nucleus, condensed chromatin, membrane blabbing and formation of apoptotic bodies in NP treated mice. Thus, the results confirmed the stimulation of apoptosis by NP Therefore, NP might render protection to macromolecules to avoid damage from xenobiotic such as DEN by maintaining the redox balance thereby exhibit anticancer activity during DEN induced liver cancer. In vivo antitumor test, pemetrexed-loaded PEG-Peptide-PCL nanoparticles exhibited best antitumor efficacy in the all the treatment groups. Similar results were observed from the studies of (Lu et al,14) in lung cancer model. Examined the antitumor effect of docetaxel-loaded PEG-Peptide-PCL nanoparticles through intravenous administration and the docetaxel-loaded nanoparticles formed by PEG-Peptide-PCL showed more efficient antitumor efficacy over PEG-PCL nanoparticles on H22 tumor-bearing mice15,16. PEG-Peptide-PCL nanoparticles contained the active peptide substrates of gelatinases and the gelatinases-responsive PEG-Peptide-PCL nanoparticles effectively targeted to the tumor tissues and cells, and exhibited distinguished antitumor potency.
In this study, we have investigated the effect of targeted drug delivery of pemetrexed on DEN/HepG2 model. Active targeting nanoparticles delivery system has become a potential new drug modality with the opportunity of enhanced water solubility, constant and stable release of drug along with minimized side effects.
In this article, we have provided positive data for antitumor and antimetastasis effect of targeted nanoparticles as a potential strategy. The model developed by us could be used in further studies. MMPs play an important role in cancer progression, including tumor growth, invasion, metastasis, and angiogenesis. For this reason, treatment methods aiming at MMPs have great significance. On the basis of this study, it concludes that the HCC model prepared was fast and equivalent to the other models present. The targeted nanoparticles prepared, has better efficacy profile with lower side effects then the free pemetrexed available in market. Low dose and high dose (20 mg/25 mg)/kg of pemetrexed treats HCC significantly. Our results provide a basis for further study of targeted nanoparticles. Our preclinical data could provide new selections and directions for future therapeutic applications.
Future perspective in this field of study could be PEG-Peptide-PCL nanoparticles represent a new therapeutic modality for clinical treatments and the pemetrexed-loaded PEG-Peptide-PCL nanoparticles may be a potent drug for inhibiting hepatic metastasis of carcinoma.
All the animal protocols were duly approved by the DIPSAR Animal Ethics Committee protocol no. IAEC/2015-I/protocol no.13 and were performed with care.
Source of funding
N.K. and S.S.A.: worked equally in this project. N.K.: research scholar; S.S.A.: instructor and mentor.
Conflict of interest disclosure
The authors declare that they have no financial conflict of interest with regard to the content of this report.
Research registration unique identifying number (UIN)
Support by Govt. of NCT of Delhi, AICTE (GPAT-scholarship), and NCCS-Pune is gratefully acknowledged.
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