Glycyrrhetinic acid-modified chitosan nanoparticles enhanced the effect of 5-fluorouracil in murine liver cancer model via regulatory T-cells.

Modified chitosan nanoparticles are a promising platform for drug, such as 5-fluorouracil (5-FU), gene, and vaccine delivery. Here, we used chitosan and hepatoma cell-specific binding molecule glycyrrhetinic acid (GA) to synthesize glycyrrhetinic acid-modified chitosan (GA-CTS). The synthetic product was confirmed by infrared spectroscopy and hydrogen nuclear magnetic resonance. By combining GA-CTS and 5-FU, we obtained a GA-CTS/5-FU nanoparticle, with a particle size of 193.7 nm, drug loading of 1.56%, and a polydispersity index of 0.003. The GA-CTS/5-FU nanoparticle provided a sustained-release system comprising three distinct phases of quick, steady, and slow release. In vitro data indicated that it had a dose- and time-dependent anticancer effect. The effective drug exposure time against hepatic cancer cells was increased in comparison with that observed with 5-FU. In vivo studies on an orthotropic liver cancer mouse model demonstrated that GA-CTS/5-FU significantly inhibited cancer cell proliferation, resulting in increased survival time. The antitumor mechanisms for GA-CTS/5-FU nanoparticle were possibly associated with an increased expression of regulatory T-cells, decreased expression of cytotoxic T-cell and natural killer cells, and reduced levels of interleukin-2 and interferon gamma.

Introduction

Drug delivery systems carry drugs to the targeted cells by exploiting the different physiological and biochemical characteristics of tumor and normal cells.1 These systems can be used to reduce the distribution and metabolism of 5-fluorouracil (5-FU) in nontarget organs and tissues. They also improve the drug efficacy and reduce side effects as a result of the lower doses that are administered. The natural polymer chitosan (CTS) and its analogs have been widely studied as drug vectors, based on their lack of toxicity, biodegradability, good biocompatibility, and absorption.2–4 Small-molecule drugs, such as 5-FU and paclitaxel, carried by CTS or its derivatives, result in extended release, improved bioavailability, and reduced side effects.5–7 These carrier agents also have adhesion and biodegradability properties, which give them the potential to improve drug efficacy. Targeted drug delivery has been shown to concentrate the drugs at the site of diseased tissue, thereby greatly reducing side effects in normal tissues and improving the biodegradability and drug efficacy.8,9 Glycyrrhetinic acid (GA) has been shown to specifically bind to receptors on the liver cell membrane, because there are more GA binding sites in hepatoma cells than in other cells.10 Therefore, when nanomaterials are synthesized with GA, they will tend to accumulate in hepatoma cells, leading to improved growth inhibition.11,12

In addition, 5-FU is a first-line anticancer drug that inhibits tumor cell proliferation by interfering with the synthesis of nucleic acid. However, its efficacy is affected by low lipophilicity and low bioavailability.13,14 In addition, its clinical use is limited by unwanted side effects, such as gastrointestinal reactions, myelosuppression, alopecia, ataxia, and its narrow therapeutic index (the therapeutic dose is close to the toxic dose).15 The structural optimization of 5-FU has been undertaken in an attempt to improve selectivity and to reduce side effects. This has resulted in the discovery of a series of prodrugs associated with fewer side effects and a higher chemotherapeutic index than 5-FU (for example, fluorouridine and tegafur, which are in clinical trials).16,17 However, the prodrugs are still limited by adverse effects resulting from the lack of selectivity for cancer cells.

The cluster of differentiation (CD)4+ CD25 regulatory T-cells (Treg) are known to modulate the immune system and to maintain tolerance to self-antigens. They also play an important role in tumor immunity and organ transplantation.18,19 Treg expression is increased in the peripheral blood and in the tumor microenvironment of patients with liver cancer and other solid tumors, and it is negatively correlated with tumor progression, prognosis, and survival rate.20,21 However, the impact of chemotherapy on Treg has yet to be established.22,23

In this study, we prepared glycyrrhetinic acid-modified chitosan (GA-CTS), which was used to synthesize a GA-CTS/5-FU nanoparticle. The results showed that the sustained-release system (GA-CTS/5-FU nanoparticle) effi-ciently targeted the drug to the liver and significantly inhibited cancer cell proliferation in an orthotropic liver cancer mouse model, resulting in an increased survival time.

Materials and methods

Mice and cell lines

The human hepatocellular carcinoma cell line (SMMC-7721 and Hep G2) and normal liver cells (LO2) were obtained from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, People’s Republic of China). The human colon cancer cell line (SW480) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and the mouse hepatoma cell line H22 was purchased from the China Center for Type Culture Collection (Wuhan, People’s Republic of China).

Female BALB/c mice, 7 weeks of age weighing about 20 g, were obtained from the Science Department of Experimental Animals of Fudan University (Shanghai, People’s Republic of China), and kept under the standard conditions of the animal room (temperature, 25°C; humidity, approximately 55%–60%) in a specific pathogen-free (level B) animal facility. All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China and were approved by the ethics committee of Pudong New Area District Zhoupu Hospital (Shanghai, People’s Republic of China) and Fudan University.

GA-CTS synthesis

The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC ⋅ HCl) (Sigma-Aldrich, St Louis, MO, USA) and N-hydroxysuccinimide (Sigma-Aldrich) were added to GA solution (1 g, Shaanxi FUJIE Pharmaceutical Co, Ltd, Xi-an City, People’s Republic of China) in dimethylformamide (AMRESCO®, Solon, OH, USA). The solution was mixed with 2% chitosan in acetic acid and stirred at room temperature. After 48 hours, the mixture was precipitated with acetone, and the precipitate was washed with 60% ethanol and ether. The final product was obtained after vacuum drying.

Fourier transform infrared spectroscopy

In addition, 85% deacetylated CTS powder (Sigma-Aldrich) and GA-CTS were pressed into a potassium bromide pellet. The composites were analyzed by Fourier transform infrared spectroscopy (NEXUS, Nicolet/Natus Medical Incorporated, San Carlos, CA, USA) in the range 4000–400 cm−1.

Hydrogen nuclear magnetic resonance experiments

To verify the structure of the CTS and GA-CTS, samples were dissolved in a solution of deuterium chloride and D2O. The hydrogen nuclear magnetic resonance (1H-NMR) spectras were recorded using a Varian NMR System 600 machine (Varian, Inc./Agilent Technologies, Santa Clara, CA, USA) at a resonance frequency of 600 MHz. Tetramethylsilane was used as a reference compound.

Preparation of GA-CTS/5-FU nanoparticles

The GA-CTS/5-FU nanoparticles were prepared by the ionic cross-linking method. Then, 5-FU was added into a CTS solution (dissolved in acetic acid) at a mass ratio of 1:1. An aqueous sodium polyphosphate (TPP) solution (0.05% w/v) was then added to the mixture and stirred. After 50 minutes, GA-CTS/5-FU nanoparticles were formed by the incorporation of the TPP solution at a ratio of GA-CTS:TPP of 5:1. The nanoparticles were centrifuged at 10,000 rpm for 30 minutes to remove NaOH and 5-FU. The product was dispersed in the double-distilled H2O for storage.

In vitro release of GA-CTS/5-FU nanoparticles

To prepare a standard curve, a series of working solutions of 5-FU (0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 mg/L) was obtained by diluting the 5-FU standard stock solution with 0.5 mL human plasma. A calibration curve was generated by linear regression of the peak area ratio of the 5-FU concentration.

GA-CTS/5-FU nanoparticles and 5-FU were placed into dialysis bags and immersed in 30 mL of simulated body fluid (SBF) at pH 7.4. The particles were incubated at 37°C and shaken at a constant speed of 60 r/m. The medium was replaced with fresh SBF at 20 minutes, 40 minutes, 1 hour, and 6 hours, and then daily for 10 days. Drug concentration in each sample was assayed in triplicate by measuring absorbance at 265 nm. Drug release and loading capacity were calculated by the following formulas:

Cytotoxicity of GA-CTS/5-FU nanoparticles

SW480, SMMC-7721, Hep G2, and LO2 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) with 10% fetal calf serum at 37°C in 5% CO2. A cell suspension containing 1 × 104 cells was plated into 96-well plates (Sigma-Aldrich) at concentrations of 190 μL per well for 24 hours. Cells were exposed to 10 μL of 5-FU or GA-CTS/5-FU nanoparticles at different doses for 24, 48, and 72 hours. Also, 30 μg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was added to each well and incubated for 4 hours. The medium was then removed; 200 μL of dimethyl sulfoxide was added. The plates were vibrated for 10 minutes. Absorbance was measured by a Bio-Rad automatic microplate reader (Bio-Rad, Hercules, CA, USA) at 490 nm. All measurements were performed in triplicate. MTT assays were performed after exposure for 4–10 days with 1.6 mg/L of 5-FU.

Cytotoxicity was determined as follows:

Orthotopic transplant mouse model of hepatocellular carcinoma

To establish a highly reproducible model of murine liver cancer, we initially inoculated BALB/c mice subcutaneously with 6 × 107 H22 cells resuspended in 100 μL of phosphate buffered saline (PBS) into the rear right flanks of the recipient animals. Once established, the tumors were subsequently isolated and digested with collagenase. This in vivo passage method excluded influences of bovine serum and culture media. After anesthesia with 20% ethyl carbamate, 6 × 107 in vivo passaged H22 cells, resus-pended in 50 μL of PBS, were subcutaneously implanted in the left lobe of the liver. The abdomen was closed after 2 minutes.

Targeting GA-CTS/5-FU nanoparticles in hepatocellular carcinoma in vivo

Following tumor implantation, the mice were randomly divided into three groups (n=5), treated with 200 μL of 5-FU, CTS/5-FU, or GA-CTS/5-FU (5-FU: 0.371 mg). The mice were sacrificed after 30 minutes. Liver tumor, liver, spleen, kidney, lung, muscle, heart, and small intestines were harvested, washed in saline solution, and dried in filter papers. The tissue (0.5–1.0 g) was homogenized, and the concentration of 5-FU in 0.5 mL of the homogenates was determined.

Inhibitory effects of GA-CTS/5-FU nanoparticles in orthotopic liver transplantation mouse model

Five days after establishment of the orthotopic liver transplantation mouse model, the tumor size reached 4–6 mm. Six days after model establishment, the mice were divided into four groups: control, GA-CTS, 5-FU, and GA-CTS/5-FU. Mice in the four groups were injected with saline, GA-CTS, 5-FU, or GA-CTS /5-FU nanoparticles, respectively, at a volume of 200 μL (all 5-FU-containing injections contained 0.371 mg of 5-FU). Drugs were administered for 5 days, and ten mice in each group were killed at 10 days by an overdose of anesthesia to harvest their tumor tissues. All efforts were made to ameliorate animal suffering. The tumor growth and tumor weight were measured in each group. The remaining 13 mice in each group were used for survival analysis.

Enzyme-linked immunosorbent assay detection of interleukin-2 and interferon-γ

Mouse serum specimens were obtained from each group and maintained at −20°C for determination of interleukin-2 (IL-2) and interferon gamma (IFN-γ). For enzyme-linked immunosorbent assay (ELISA) (Santa Cruz Biotechnology, CA, USA) detection, serum specimens were thawed at 37°C in an incubator and diluted with double-distilled H2O GA-CTS to a volume of 500 mL. The IL-2 and IFN-γ standards were diluted to 8,000 µg/L. The diluted specimens (150 μL) or standards (50 μL) were added to 96-well plates for 15 minutes. They were then oscillated and incubated for 2 hours at room temperature. The solution in the wells was removed, and the wells were washed four times with 400 μL detergent, prior to the addition of horseradish peroxidase IL-2 and IFN-γ (200 µL). The cells were then oscillated and incubated for 2 hours at room temperature. The liquid was removed and was washed another four times with 400 µL detergent. Equal amounts of developer A and B were mixed, added to the 200 μL enzyme-labeled antibody, and incubated at room temperature in the dark for 30 minutes. Stop buffer (50 μL) was then added to end the enzyme reaction. The specimens were immediately analyzed, and optical density was assessed at a wavelength of 450 nm. All samples and standards were run in triplicate. The assay sensitivity for IL-2 and IFN-γ was 0.1 units/mL.

Flow cytometry

Mice spleens were harvested, carefully ground up, and filtered using mesh screens to obtain single cell suspensions, which were incubated in RPMI 1640 culture solution. The resulting cells were seeded into a 96-well staining plate and centrifuged at 300× g for 5 minutes. The cell pellet was washed two to three times in 200 μL and incubated for 1 hour at 4°C with a blocking agent under gentle shaking. The cells were then washed two to three times with 200 μL PBS. Diluted antibodies (for example, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD25 antibody, anti-CD19 antibody, anti-CD11b antibody, anti-CD27 antibody, and anti-Foxp3 antibody from BD Bioscience, San Jose, CA, USA) were added to the corresponding wells and incubated for 1 hour at 4°C with gentle shaking. The cells were washed again with 200 μL PBS and resuspended in 200 μL cold PBS in the dark for the determination of total T-cells (CD3+), T-helper cells (phenotype CD3+CD4+), cytotoxic T-cell (CTL) (phenotype CD3+CD8+), and B-cells that expressed CD3-CD19+. The assay also included natural killer (NK) cells that expressed CD11b+CD27+ and Treg cells that expressed CD4+CD25+Foxp3+. All cell assays were conducted using flow cytometry (BD, Franklin Lakes, NJ, USA).

MTT detection of cytotoxic T-cell and NK cell activity

Spleen cells (NK cells and cytotoxic T-cells [CTL]) were selected by flow cytometry, and their concentrations were adjusted to 1 × 106 cells/mL. Lymphoma cell line (YAC-1) and H22 were used as target cells for NK and CTL reactivity. The cells were incubated in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% CO2 for 48 hours. The cell densities were adjusted to 1 × 105 cells/mL.

Three mice were randomly selected for detection of CTL and NK cell activity. Spleen and target cells (100 μL at an effector:target cell ratio of 10:1) were added to 96-well staining plates. After 18 hours of incubation, MTT (20 μL) was added to each well of the plate. Incubation continued for 4 hours. After horizontal centrifugation, supernatant in the plates was removed. Dimethyl sulfox-ide (200 μL) was added to the plates and oscillated for 20 minutes. Absorbance was determined at a wavelength of 570 nm (A570). The activity of CTL and NK cells were calculated as:

Statistical analysis

All data were expressed as means ± standard deviations (SD). One-way analysis of variance and the least signifi-cance difference test were used for intergroup comparisons. Kaplan–Meier survival plots were used to analyze the survival data. In all analyses, values of P,0.05 were considered statistically significant.

Results

Infrared spectra and 1H-NMR of GA-CTS

Figure 1A shows the infrared (IR) spectra of GA, CTS, and GA-CTS. The IR spectra of GA existed as an oleanane-type pentacyclic triterpene skeleton. The characteristic absorption peak was at the bending vibration absorption of the C–H in-plane group of the gem-dimethyl moiety connected to C4. The characteristic absorption peak of p-π conjugated carbonyl group of C11 was at 1,664 cm−1. It existed as a C30 carboxyl group with an absorption peak at 1,706 cm−1. The peak at 3,440 cm−1 represented the stretch vibration absorption peak of C3. The IR spectra of the CTS showed three characteristic amide absorption peaks: an amide band I at 1,655 cm−1, an amide band II at 1,600 cm−1, and an amide band III at 1,323 cm−1. The relatively weak absorption peak of amide band I and the relatively strong absorption peak of amide band II indicates that there was a high degree of deacetylation of the CTS. Characteristic absorption peaks of carbohydrates were observed at 1,155 cm−1 for the asymmetric stretching vibration absorption peak at C–O–C and at 1,078 cm−1 and 1,025 cm−1 for the skeleton-stretching vibration absorption peak of C–O. The stretching vibration absorption peak of O–H and N–H was represented by the wave at 3,440 cm−1 and by the stretching vibration peak at 2,877 cm−1. Absorption bands shifts for GA-CTS were seen at 1,655 cm−1 of amide band I, 1,600 cm−1 of amide band II, and 1,323 cm−1 of amide band III in CTS, and at 1,654 cm−1, 1,560 cm−1, and 1,314 cm−1, respectively, in the GA-CTS nanoparticles. The intensity of amide band I increased, while the intensity of the amide band II decreased. The absorption band of carboxyl group in GA (1,706 cm−1) disappeared. These changes were attributed to the formation of the amide bond between the GA carboxyl group and the CTS amine.

Figure 1

IR spectra and 1H-NMR of GA-CTS.

Notes: (A) The mass spectrum of the GA-CTS, amide bond was formed by the carboxylic acid group of GA and the amino group of CTS. (B) 1H-NMR of GA-CTS showing that GA was connected to CTS. (C) Synthetic scheme for GA-CTS.

Abbreviations: GA-CTS, glycyrrhetinic acid-modified chitosan; CTS, chitosan; GA, glycyrrhetinic acid; IR, infrared; 1H-NMR, hydrogen nuclear magnetic resonance; EDC ⋅HCl, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

Figure 1B shows 1H-NMR results of CTS and GA-CTS. Chemical shifts of 1.410 ppm and strong characteristic absorption peaks at 0.566 were observed for CTS, which were attributed to the protons in the −CH3, −CH2, and −CH groups of GA. These data indicate the successful connection of GA to the amino group of CTS.

Characterization of GA-CTS/5-FU nanoparticles

Scanning electron microscopy of GA-CTS/5-FU showed the presence of spherical nanoparticles (size 193.7 nm) with smooth surfaces (Figure 2A). The polydispersity index (PI) was 0.003, indicating good dispersion (Figure 2B). The zeta potential was +27.4 mV; drug loading efficiency was 1.56%.

Figure 2

SEM chart and characteristics of the GA-CTS/5-FU nanoparticle.

Notes: (A) The particles were spherical with a smooth surface, and there was no adhesion between nanoparticles. (B) Particle size graph showing the diameter of GA-CTS/5-FU (193.7 nm). (C) The in vitro release curve of nanoparticles in simulated body fluid (37°C at pH 7.4). Data are mean ± SD (n=3).

Abbreviations: GA-CTS, glycyrrhetinic acid-modified chitosan; 5-FU, 5-fluorouracil; SD, standard deviation; SEM, scanning electron microscopy.

To study the release of GA-CTS/5-FU nanoparticles, an in vitro release curve was generated using simulated body fluid. Linear regression of the peak area ratio of the 5-FU concentration (0.1 to 20 mg/L) curve was calculated as:

Rapid release was observed for 5-FU SBF within a cumulative release percentage of 95.7% within 1 hour. Nanoparticle release occurred in three stages (Figure 2C). Rapid release was observed between 0–6 hours, which was followed by a cumulative release of 20.6% due to the diffusion of surface 5-FU into the SBF solution. A smooth slow-release process occurred between 6 hours and Day 7, resulting in a cumulative release of 91.4%. From Day 7 to Day 10, the release reached a plateau, with a cumulative release percentage of 95.6%. There was an additional release of only 4.2% at Day 10.

Dose- and time-dependent cytotoxicity of GA-CTS nanoparticles in tumor cells

As shown in Figure 3A, GA-CTS/5-FU and 5-FU inhibited the growth of tumor cells in a dose-dependent manner. At 24 hours, 48 hours, and 72 hours, cancer cell proliferation inhibition rates were the highest for GA-CTS/5-FU in Hep G2 cells, followed by GA-CTS/5-FU in SMMC-7721 cells, 5-FU in Hep G2 cells, 5-FU in SW480 cells, 5-FU in SMMC-7721 cells, GA-CTS/5-FU in SW480 cells, GA-CTS/5-FU in LO2, and 5-FU in LO2, indicating a maximum inhibition of hepatic cancer cells by GA-CTS/5-FU (P<0.01). Between Day 1 and Day 5, the inhibition rate for GA-CTS/5-FU in SW480 was lower than for 5-FU in SW480, SMMC-7721, and Hep G2 cells. By contrast, the cytotoxic effect of GA-CTS/5-FU in SW480 was higher Day 6 to Day 10, possibly indicating the release of GA-CTS/5-FU nanoparticles and resulting in a longer duration of action (Figure 3B).

Figure 3

Inhibition rates of GA-CTS/5-FU and 5-FU in SW480, SMMC-7721, Hep G2 and LO2 cells detected by MTT assays.

Notes: (A) Inhibition rates of different doses of 5-FU on SW480, SMMC-7721, Hep G2, and LO2 cells at 24, 48, and 72 hours. (B) Inhibition rates of different groups on SW480, SMMC-7721, and Hep G2 cells after 1 to 10 days (5-FU: 1.6 mg/L). Data are mean ± SD (n=3).

Abbreviations: 5-FU, 5-fluorouracil; GA-CTS, glycyrrhetinic acid-modified chitosan; LO2, normal liver cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation.

In vivo antitumor efficacy of GA-CTS/5-FU

As shown in Figure 4A–C, tumor weights and volumes were significantly lower in the GA-CTS/5-FU group than in the GA-CTS or control groups (P<0.01). In the 5-FU group, tumor weight and volume were significantly lower than in the control group (P<0.01), but no differences were seen between the GA-CTS group and the controls (P>0.05).

Figure 4

Effect of GA-CTS/5-FU on orthotopic cancer liver model in mice.

Notes: On Day 5, mice were treated with GA-CTS/5-FU, 5-FU, GA-CTS, and PBS. After 10 days, the mice were sacrificed and tumor weight and volume were observed. Results are mean ± SD (n=10). (A) Tumor weight; (B) tumor volume; (C) general picture of tumor tissue; (D) Kaplan–Meier analysis indicated that median survival was highest for GA-CTS/5-FU, followed by 5-FU, GA-CTS, and control group. **P<0.01 compared with control or GA-CTS; ##P,0.01 compared with 5-FU.

Abbreviations: GA-CTS, glycyrrhetinic acid-modified chitosan; 5-FU, 5-fluorouracil; PBS, phosphate buffered saline; SD, standard deviation.

A Kaplan–Meier analysis showed that the median survival was 16 days in the control group, with all the mice dying between Day 11 and Day 20 (Figure 4D). Mice in the GA-CTS group had a median survival of 17 days, with all the mice dying between Day 12 and Day 22. Mice died between Day 18 and Day 30 in the 5-FU group, with a median survival of 24 days. Mice treated with GA-CTS/5-FU had a significantly longer median survival (35 days) than in other groups (P<0.01), with all mice dying between Day 21 and Day 43.

Effect of GA-CTS/5-FU on expression of IL-2 and IFN-γ in mice

As shown in Figure 5, IL-2 and IFN-γ levels were 82.34±7.55 pg/mL and 987.54±78.48 pg/mL in the GA-CTS/5-FU group, 54.57±3.82 and 415.20±86.34 pg/mL in the 5-FU group, 111.08±9.64 and 1352.80±80.61 pg/mL in the GA-CTS group, and 82.87±6.19 and 1057.80±82.81 pg/mL in the control group (P<0.01). Thus, IL-2 and IFN-γ levels significantly decreased after treatment with 5-FU; they significantly increased when treated with GA-CTS. There was no difference in IL-2 and IFN-γ levels between the GA-CTS/5-FU and the control group, although values were significantly higher in the GA-CTS/5-FU than the 5-FU group (P<0.01).

Figure 5

Effect of GA-CTS/5-FU on the expression of IL-2 and IFN-γ GA-CTS/5-FU in an orthotopic liver transplantation model in mice.

Notes: Mice were treated with GA-CTS/5-FU, 5-FU, GA-CTS, and PBS. Blood was collected after 10 days. (A) Concentration of IL-2 in serum on Day 10 determined by ELISA (n=3). (B) Concentration of IFN-γ in serum on Day 10 determined by ELISA (n=3). Results are mean ± SD. **P<0.01 compared with control. ##P<0.01 compared with 5-FU.

Abbreviations: IL, interleukin; IFN-γ, interferon-γ; GA-CTS, glycyrrhetinic acid-modified chitosan; 5-FU, 5-fluorouracil; PBS, phosphate buffered saline; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.

Effect of GA-CTS/5-FU on NK, CTL, and Treg cells

Spleen cells were extracted for flow cytometry and MTT assays to explore the mechanism of the antitumor effect of GA-CTS/5-FU in the orthotopic liver transplantation model. There was no difference in the percentage of CD3-CD19+, CD3+CD4+, and CD3+ cells after treatment (Figure 6G, P>0.05). Significantly reduced percentages of CTL and NK cells and significantly reduced cytotoxicity were observed after the 5-FU treatment (P<0.01). These changes were all reversed in the GA-CTS group (P<0.01). No difference was observed between the GA-CTS/5-FU and the control group. However, the percentage of CTL and NK cells and the degree of cytotoxicity were significantly higher than in the 5-FU group (P<0.01) (Figure 6A, B, D, E, and H). These data indicate that the antitumor mechanism for the GA-CTS/5-FU nanoparticles may be associated with the decreased percentages of CTL and NK cells.

Figure 6

Effect of GA-CTS/5-FU on NK, CTL, and Treg cells.

Notes: Mice were treated with GA-CTS/5-FU, 5-FU, GA-CTS, and PBS. After 10 days, the mice were sacrificed. Spleen cells were extracted for flow cytometry and MTT assays. Flow chart showing the percentage of (A) NK cells, (B) CTL cells, (C) and Treg cells. Histogram of the percentage of (D) NK cells, (E) CTL cells, (F) Treg cells, (G) T cells, Th cells, and B cells, (H) Cytotoxicity on NK and CTL cells was determined by MTT assays. (n=3). Results shown as mean ± SD. **P<0.01 compared with control. ##P<0.01 compared with 5-FU.

Abbreviations: GA-CTS, glycyrrhetinic acid-modified chitosan; 5-FU, 5-fluorouracil; PBS, phosphate buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NK, natural killer; CTL, cytotoxic T-cell; Treg, regulatory T-cell; SD, standard deviation; FSC forward scatter; H, high; Th, T helper.

Tregs are a subpopulation of T-cells that modulate the immune system. Tregs come in many forms, the most well-understood being those that express CD4+CD25+ Tregs. Our results demonstrated that the percentage of Treg cells in the spleen significantly increased after the treatment with 5-FU (P<0.01) and significantly decreased after treatment with GA-CTS (P<0.01). No change in the Treg cells was observed in the GA-CTS/5-FU group (P>0.05); percentages were significantly lower than in the 5-FU group (P<0.01) (Figure 6C and F). These results suggest that the antitumor mechanism for the GA-CTS/5-FU nanoparticle may be associated with the decreased percentage Treg cells.

Discussion

Targeted drug therapy can specifically deliver drugs to tumor tissues, resulting in increased local concentrations and reduced toxicity. GA, an aglycone of glycyrrhizin, acts as an antioxidant and detoxifying agent. It has been shown to increase apoptosis in hepatoma cells.24,25 It also has the ability to target the liver26,27 and has been shown to specifically bind to receptors on the liver cell membrane.28 These properties make GA a suitable candidate for use in the development of novel drugs that target liver disease (Figure 1C).

In the present study, GA-CTS, identified by IR and 1H-NMR, was used to investigate the feasibility of using GA-CTS as a drug carrier to the liver. In addition, 5-FU is a widely used anticancer drug that is toxic to normal cells and has a short plasma half-life of 15–20 minutes. Improving the bio-availability and site-specific delivery of 5-FU and reducing its side effects may provide obvious therapeutic advantages. In this study, we prepared GA-CTS/5-FU nanoparticles with an average diameter of 193.7 nm. The scanning electron microscopy imaging showed that GA-CTS/5-FU nanoparticles were spherical, smooth surface structures that adhered to one another and were not fully dispersed after centrifugation. The PI is an important indicator of the physical stability of nanoparticles. PI values between 0.1 and 0.25 indicate good uniformity, while values >0.5 are indicative of poor uniformity.29 Prior to the centrifugation, the GA-CTS/5-FU nanoparticles used in our study had a PI value of 0.003, with a single peak, indicating good uniformity. The release of nanoparticles in SBF occurred in stages, characterized by a sustained release, which was similar to previous studies.30 Rapid release was observed between 0–6 hours, with a cumulative release of 20.6%, resulting from the diffusion of surface 5-FU into the SBF solution. Smooth, slow release occurred between 6 hours and 7 days. Following the gradual degradation of insoluble hydrophobic material, the drug diffused through the membrane, resulting in a cumulative release of 60.8%. Between Day 7 and Day 10, there was a residual drug release of 4.2% on Day 10.

The antitumor mechanism of 5-FU is associated with the metabolite 5-fluorodeoxyuridine monophosphate which inhibits the thymidylate synthase activity and blocks the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate which, in turn, affects DNA synthesis.31,32 Between Day 1 and Day 5, the inhibition for GA-CTS/5-FU in SW480 was lower than 5-FU, probably due to the slow release of 5-FU encapsulated by GA-CTS and the low cellular concentrations of 5-FU. The inhibition reached a plateau between Day 6 and Day 10. This profile was not observed with the GA-CTS nanoparticle group, which had a longer duration of action. These findings are in general agreement with our previous report demonstrating the inhibition of tumor cells by galactosyl chitosan/5-FU.33

Our results also demonstrated that tumor weight and volume in a mouse orthotopic liver transplantation model were significantly lower in animals who had received the GA-CTS/5-FU than in other groups. No antitumor effect was observed in the GA-CTS group. The Kaplan–Meier analysis showed a longer median survival in the GA-CTS/5-FU than in the 5-FU group, further indicating better antitumor efficacy. This suggests that GA binding might result in a higher concentration of 5-FU in the hepatoma cells.34

GA-CTS/5-FU also attenuated the toxicity caused by the 5-FU. These results are similar to those reported by Tian et al.11 In this paper, the 5-FU on liver and liver cancer cells has an obvious inhibitory effect; the inhibition rates of liver cancer cells were stronger than in the liver cells.11 The argument of the 5-FU by GA-CTS on an inhibition of liver cancer cells was significantly stronger than that of the liver cells. Its mechanism may be significantly related to the fact that the GA receptors expressed on the surface of cancer cells are more obvious than those on the surface of liver cells.10

To explore the mechanism of the GA-CTS/5-FU nanoparticle, we determined the IL-2 and IFN-γ levels in the serum by ELISA. The activity of NK and CTL cells from the spleen were evaluated, using MTT assays, and the percentage of CTL, NK cells, and Treg were determined by flow cytometry. Immunosuppression was observed in the mice treated with 5-FU, evidenced by the reduced levels of IL-2 and IFN-γ, by decreased percentages of CTL and NK cells, and by an increase in the percentage of Treg cells. The GA-CTS/5-FU nanoparticle improved immune function. It increased IL-2 and IFN-γ levels, decreased percentage of Treg, and increased the percentages of CTL and NK cells. The glycoprotein IFN-γ is mainly secreted by CTL and NK cells and is reported to have antiviral, immunomodulatory, and antiproliferative properties.35,36 The IFN-γ inhibits tumor cells and has immunomodulatory activity. It has also been shown to activate and to improve phagocytosis in monocyte, macrophage, and NK cells, and favorably alters the differentiation and maturation profile of the B-cell and T-cell. Other studies have shown that IFN-γ enhances the cytotoxicity of CTL cells and induces cellular expression of the major his-tocompatibility complex class I antigen, which improves the immune recognition of CTL and NK cells.37 IL-2 is a growth factor of the T lymphocytes. It plays an important role in the activation and proliferation of T lymphocytes and in the activation of B-cells and macrophages. Conversely, IL-2 is also secreted by activated T-cells.38 Tregs are a subpopulation of T-cells that modulate the immune system and play an important role in tumor immunity. Foxp3 is a specific transcription factor that is essential for the development and function of Treg cells.39 Treg cells inhibit the activation and proliferation of CTL cells, resulting in immunosuppression.40 Therefore, the mechanism by which the GA-CTS enhanced the antitumor activity of 5-FU may be related to attenuated immunosuppression, leading to an increase in the population of Treg cells and a decrease in the population of CTL and NK cells, coupled with reduced levels of IL-2 and IFN-γ.