Preparation of T‑2 toxin‑containing pH‑sensitive liposome and its antitumor activity

  • Authors:
    • Yuan Dong
    • Guixian Meng
    • Jian Guo
    • Moli Yin
    • Huijing Xu
    • Yujie Li
    • Jie Zhu
    • Wenhe Zhu
    • Mingguang Li
    • Yan Li
    • Huiyan Wang
  • View Affiliations

  • Published online on: September 22, 2020     https://doi.org/10.3892/mmr.2020.11531
  • Pages: 4423-4431
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Abstract

T‑2 toxin is a type A trichothecene mycotoxin. In order to reduce the side effects of T‑2 toxin and increase the tumor targeting ability, a pH‑sensitive liposome of T‑2 toxin (LP‑pHS‑T2) was prepared and characterized in the present study. The cytotoxicity of LP‑pHS‑T2 on A549, Hep‑G2, MKN‑45, K562 and L929 cell lines was tested by 3‑(4,5‑dimethylthiazolyl‑2)‑2,5‑diphenyltetrazolium bromide assay, with T‑2 toxin as the control. The apoptotic and migratory effects of LP‑pHS‑T2 on Hep‑G2 cells were investigated. The preparation process of LP‑pHS‑T2 involved the following parameters: Dipalmitoyl phosphatidylcholine: dioleoylphosphatidylethanolamine, 1:2; total phospholipid concentration, 20 mg/ml; phospholipid:cholesterol, 3:1; 4‑(2‑hydroxyethyl)‑1‑piperazineethanesulfonic acid buffer (pH 7.4), 10 ml; drug:lipid ratio, 2:1; followed by ultrasound for 10 min and extrusion. The encapsulation efficiency reached 95±2.43%. The average particle size of LP‑pHS‑T2 after extrusion was 100 nm; transmission electron microscopy showed that the shape of LP‑pHS‑T2 was round or oval and of uniform size. The release profile demonstrated a two‑phase downward trend, with fast leakage of T‑2 toxin in the first 6 h (~20% released), followed by sustained release up to 48 h (~46% released). From 48‑72 h, the leakage rate increased (~76% released), until reaching a minimum at 72 h. When LP‑pHS‑T2 was immersed in 0.2 mol/l disodium phosphate‑sodium dihydrogen phosphate buffers (pH 6.5), the release speed was significantly increased and the release rate reached 91.2%, demonstrating strong pH sensitivity. Overall, antitumor tests showed that LP‑pHS‑T2 could promote the apoptosis and inhibit the migration of Hep‑G2 cells. The present study provided a new approach for the development of T‑2 toxin‑based anti‑cancer drugs.

Introduction

T-2 toxin (4β,15-diacetoxy-8α-(3-methylbutyryloxy)-3α-hydroxy-12, 13-epoxytrichothece-9-ene; C24H34O9) (relative molecular weight 466.52), a type A trichothecene mycotoxin, is the secondary metabolite of Fusarium sporotrichioides and F. Langsethiae (1,2). T-2 toxin is found extensively in moldy cereals (wheat, maize, barley and oats) and moldy food (3). It is acknowledged as an unavoidable contaminant in human foods (4). Exposure to T-2 toxin causes oral injury (5), liver injury (6), decrease in body weight, gastrointestinal injury and even mortality (7). The severe tissue damage caused by T-2 toxin is associated with the inhibition of protein and DNA synthesis, metabolic alteration, cell membrane injury, immunosuppression, and glycoprotein and collagen synthesis (810), thus resulting in apoptosis. T-2 toxin poses great harm to the health of human beings and livestock.

Early studies on T-2 toxin only focused on toxicology and metabolism aspects (1113). In the 20th century, it was reported that T-2 toxin has toxic effects on a number of cancer cell types (14). T-2 toxin can induce apoptosis in HL-60 promyelotic leukemia cells and hepatocellular carcinoma cells (15). The mechanism of apoptosis induced by T-2 toxin is proposed to be linked with oxidative stress and the activation of caspase 3/9 and the mitochondrial pathway (16,17). Therefore, the toxin possesses potential applications in tumor treatment. However, due to the toxicity of T-2 toxin on normal cells, T-2 by itself does not exhibit selectivity for tumoral tissues and hence might be characterized by a low therapeutic index. A search for an alternative strategy is required.

The use of targeted drugs is a valuable method to solve the aforementioned selectivity issues (18). Liposomes are phospholipid bilayer vesicles that possess great potential for application in the targeted delivery of chemotherapeutics in the treatment of cancer (19). The use of liposomes as drug carriers for chemotherapeutic targeting to tumor tissues is based on their greater advantages compared with other dosage methods, due to their low systemic toxicity, bioavailability and the capability to enhance the solubility of a range of chemotherapeutic agents, in addition to their ability for encapsulation of hydrophilic and lipophilic drugs (20). Liposomes can reduce drug toxicity without changing drug efficacy against tumor cells, making them a highly efficient targeting drug carrier (21). Liposomes enhance the anticancer drug therapeutic index by increasing the drug concentration in tumor cells through tumor targeting (22). The most advanced targeting strategies proposed for treating cancer involve the development of multifunctional liposomes, with combined targeting mechanisms. There are a number of types of targeting strategies for liposomes, such as temperature-, light-, redox reagent- and pH-sensitive (2325). Due to their characteristics of targeting the acidic tumor microenvironment (pH 6.8–6.5), pH-sensitive liposomes have received much attention recently (26,27). pH-sensitive liposomes consist of phosphatidylcholine or dioleoylphosphatidylethanolamine (DOPE), which are stable at physiological pH (pH 7.4), but undergo destabilization under acidic conditions (28). Various antitumor drug-containing pH-sensitive liposomes have been successfully prepared, such as those for 5-fluorouracil, doxorubicin (DOX) and taxol (2931).

The objective of the present study was to design and optimize the preparation process of a novel pH-sensitive liposomal delivery system containing T-2 toxin (LP-pHS-T2). The particle size, stability and pH-sensitivity of the liposomes in buffers were determined. Furthermore, the antitumor activity of pHS-LP-T2 was evaluated in vitro. This is an exploration of T-2 toxin as a new antitumor drug.

Materials and methods

Reagents

1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG-2000), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), DOPE and cholesterol (chol) were purchased from Shanghai Dongshang Biotechnology Co., Ltd. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, propidium iodide (PI), NaCl, HCl, high performance liquid chromatography (HPLC) grade methanol and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) sodium salt were purchased from Thermo Fisher Scientific, Inc. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc.

T-2 toxin was separated and purified from corn contaminated with Fusarium sporotrichioides by graduated organic solvent extraction, silica column chromatography and preparative HPLC, as previously described (32). Purity, >98%, the three-dimensional HPLC chromatogram of T-2 toxin is shown in Fig. S1.

The A549, Hep-G2, MKN-45 and K562 cell lines are human tumor cell lines and were kept at −80°C; L929 cells are mouse fibroblast cells (normal cells) and were kept at −80°C. All cell lines were purchased from the American Type Culture Collection. All cells were incubated in DMEM with 10% FBS and 1% antibiotics in a humidified atmosphere containing 5% CO2/95% air at 37°C. The Hep-G2 cells were authenticated as a hepatoma cell line using short tandem repeats (STR) profiling.

HPLC analysis of T-2 toxin

The concentration of T-2 toxin was determined using a HPLC system (Waters Corporation) equipped with an Agilent ZORBAX SB-C3 column (250×4.6 mm, 5 µm; Agilent Technologies Inc.). The mobile phase was methanol/water (60:40, v/v) driven by a double pump (Waters 150; Waters Corporation) at a flow rate of 1 ml/min. The amount of T-2 toxin was detected at absorption wavelengths of 198 nm with an injection volume of 10 µl at 30°C. Each sample was spiked with 6 ng/ml T-2 toxin as the internal standard. Each run was performed in triplicate. T-2 toxin limit of detection was determined by dissolving T-2 toxin at decreasing concentrations in methanol until the signal-to-noise ratio was equal to 3. According to the previous methods (33), the linearity of the standard curves, intraday and interday precision, and accuracy were determined using six T-2 toxin concentrations of 100, 200, 400, 600, 800 and 1,000 µg/ml.

Preparation of liposomes

Due to the hydrophobicity of T-2 toxin and its high solubility in methanol, T-2 toxin was wrapped in the lipid bilayer of the liposome. T-2 toxin-loaded pH-sensitive liposomes were prepared using the thin-film hydration method (34). In brief, T-2 toxin or DPPC:DOPE:chol at 1:2:1 (weight:weight:weight) was dissolved in ethanol, respectively. T-2 toxin and phospholipid mixture were mixed in a ratio of 1:1, 1:2, 1:3, 2:1, 4:1, 5:1, 6:1 or 10:1 and a total phospholipid concentration of 5, 10, 15, 20 or 25 mg/ml, respectively. The ethanol was then removed using a rotary evaporator at 40°C, until a uniform film was formed at the bottom of the flask. The film was hydrated with an appropriate volume of 20 mM HEPES buffer solution for 1 h. Liposomes were sonicated with a 20-kHz frequency probe-type sonicator (Xinhi Biolab Co., Ltd.) at 300 W for 10 min with 5-sec intervals in an ice bath. After ultrasonication, titanium particles released from the probe were removed by centrifugation at 2,000 × g for 10 min at room temperature. Free T-2 toxin was removed by ultrafiltration with a 300 K membrane filter (pore size 0.2 µm; Sartorius Stedim Biotech; Sartorius AG) at 6,000 × g for 30 min at room temperature. Finally, the liposomes were filtered through a NanoAble-150 Extruder (PhD Technology LLC) equipped with a 200-µm pores of polycarbonate membrane three times. An equal volume of 60% methanol was added to the liposomes to release T-2 toxin from the inside of the liposomes. The amount of released T-2 toxin in the liposomes was determined by HPLC and the entrapment efficiency of LP-pHS-T2 was calculated according to the following equation: EE (%) = Wencapsulated/Wtotal ×100, where EE is entrapment efficiency, Wtotal is the total amount of T-2 toxin initially added in the liposome preparation and Wencapsulated is the amount of T-2 toxin encapsulated into the liposomes.

Particle size and ζ potential

The particle diameter, polydispersity index (PDI) value and ζ potential of LP-pHS-T2 were measured by laser light scattering using a particle size analyzer according to the manufacturer's protocol (Zetasizer 3000HSA; Malvern Instruments, Ltd.) (35). The determination was repeated three times for each sample.

Morphology

The morphology of the LP-pHS-T2 was observed using a transmission electron microscope (TEM; FEI; Thermo Fisher Scientific, Inc.). For TEM studies, the LP-pHS-T2 was two-fold diluted with deionized water, and the final dilution of 0.25 mg/ml was placed on the surface of a copper grid. Next, 2% aqueous solution of sodium phosphotungstate was added for negative staining at room temperature for 15 min. Following air-drying, the copper grid was placed in the TEM (magnification, ×2,000) and imaged using Gatan DigitalMicrograph software version 1.4.3 (Gatan, Inc.) (35).

Drug release profile in vitro

The release of T-2 toxin from LP-pHS-T2 in vitro was monitored using a dialysis method (35). LP-pHS-T2 (1 ml) was added into a dialysis bag with a molecular weight cutoff of 6,000-8,000 Da and immersed in 20 ml phosphate-buffered solution (pH 7.4) at 37°C for 0, 1, 2, 4, 8, 12, 24, 48 or 72 h. An equal volume of 60% methanol was added to the liposomes. The amount of T-2 toxin released at each time point was determined by HPLC.

Stability of LP-pHS-T2 at different pH values

LP-pHS-T2 (1 ml) was added into a 6,000 to 8,000-Da molecular weight-cutoff dialysis bag and immersed in 1/15 mol/l disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 5) or a series of 0.2 mol/l disodium phosphate-sodium dihydrogen phosphate buffers (pH 5, 5.5, 6, 6.5 and 7) at 37°C for 30 min. An equal volume of 60% methanol was added to the liposomes. The amount of T-2 toxin released was determined by HPLC as aforementioned.

Antitumor activity of LP-pHS-T2
Cytotoxicity assay

MTT assay was used to evaluate the cytotoxicity of T-2 or LP-pHS-T2 on A549, Hep-G2, MKN-45, K562 and L929 cell lines (36). The cells were seeded on 96-well culture plates at a density of 5×105 cells/well. Following incubation overnight, fresh medium containing T-2 toxin or LP-pHS-T2 at final concentrations of 0.5, 5, 10 and 15 µg/ml was added to the cells at 37°C. After incubation for 48 h at 37°C, the plates were washed with PBS and incubated with 5 mg/ml MTT for 4 h at 37°C in darkness. The supernatant was aspirated and 100 µl DMSO was added to each well to dissolve the purple formazan crystals. After continuous agitation for 15 min, the reaction product was quantified by measuring the absorbance at 495 nm using a Multi-plate Reader (Model 680; Bio-Rad Laboratories Inc.). The IC50 values for each cell line were calculated using the GraphPad prism 7.0 software (GraphPad Software, Inc.) and compared between T-2 toxin and LP-pHS-T2.

Apoptosis detection using Hoechst staining

Hep-G2 cells were randomly selected from the four tumor cell lines and cultured in six-well plates for 24 h at 37°C, with a density of 5×105 cells/well. The cells were treated with 10 µg/ml of T-2 or LP-pHS-T2 at 37°C. After 4 h of incubation, the cells were fixed with 4% paraformaldehyde at 37°C, followed by staining with Hoechst 33342 to stain the nucleus for 30 min at 37°C in darkness (36). Cell imaging was performed with a fluorescence inverted microscope combined with cellSens standard version 1.5 software (magnification, ×20; IX70; Olympus Corporation). Areas of cells stained with blue fluorescence were imaged. Each group was images three times and the picture with the most stained cells was selected.

Migration assay

The Hep-G2 cells were plated in 6-well plates, cultured to 100% confluence at 37°C for 24 h, and then scratched with a p200 pipette tip (diameter, 0.57 mm). The plates were washed with PBS three times. Fresh serum-free medium containing 10 µg/ml T-2 toxin or LP-pHS-T2 was added to the cells. After 24-h culture, the distances of migrating cells were analyzed to evaluate the cell migratory ability by a fluorescence inverted microscope combined with cellSens standard version 1.5 software (magnification, ×20; IX70; Olympus Corporation), which was performed as previously described (37). The average width of the wound was measured.

Apoptosis detection via flow cytometry

The Hep-G2 cells (5×105 cells/sample) were incubated in 6-well plates at 37°C. T-2 or LP-pHS-T2 (10 µg/ml) was added to the cells, which were incubated at 37°C for 18 h. Apoptosis was measured by a PI/Annexin V-FITC dual-staining kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols, and a BD Accuri™ C6 flow cytometer (BD Biosciences) combined with cFlow version 1.023.1 software, as previously described (38). Each assay was repeated in triplicate.

Statistical analysis

All data were analyzed by Origin 8.0 (OriginLab Corporation) and are presented as the mean ± standard deviation. All data were measured in triplicate. The results were in normal distribution. The statistical significance among multiple groups was evaluated using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

T-2 toxin detection using HPLC

HPLC was used to determine the concentration of T-2 toxin. The standard curve, the intraday precision, the interday precision, the accuracy and the limit of detection were investigated (Table I); the linear regression equation was y=11393.41×+14937.51 (where × = concentration of T-2 toxin in µg/ml and y = the peak area) and the coefficient of determination was R2=0.999, indicating good linearity. Intraday (n=3) and interday (n=3) precision was not >10% in any of the assays. The limit of detection was 14.78±0.85 µg/ml.

Table I.

Establishment of T-2 toxin detection.

Table I.

Establishment of T-2 toxin detection.

Detection parameterValue
RSD, %
  Intraday precision1.42±0.14
  Interday precision1.94±0.58
  Accuracy2.93±0.15
Limit of detection, µg/ml14.78±0.85
Preparation and characterization of liposomes
Optimization of liposome preparation process by a single factor experiment

On the basis of preliminary experiments, the effects of six influencing factors [type of phospholipid, DPPC:DOPE (w/w), phospholipids:chol (w/w), hydration volume, phospholipid concentration and drug-lipid ratio] on EE values were investigated by a single factor experiment. As presented in Fig. 1A, several phospholipids commonly used in the preparation of pH-sensitive liposomes, including DOTAP, DOPE, DPPC, DSPE-mPEG-2000 and DOPC, were investigated. DOPE and DPPC were the most efficient phospholipids for encapsulating T-2 toxins, hence were chosen for the following experiments. Similarly, the highest EE values was obtained when the T-2 toxin or DPPC:DOPE:chol=1:2:1 (w/w/w) were mixed at the total phospholipid concentration of 20 mg/ml and drug-lipid ratio of 2:1 (w/w) and hydrated with 10 ml of 20 mM HEPES buffer solution as shown in Fig. 1B-F. Next, after sonication and extrusion, the maximum EE was 95±2.43%.

Characterization of LP-pHS-T2

The mean particle size of LP-pHS-T2 was ~267 nm before extrusion (Fig. 2A) and 100 nm after extrusion (Fig. 2B). These nanoparticles ranging from 100–150 nm possess advantages in controllable pore diameter and biocompatibility (39). Data from the particle size analyzer showed that the ζ potential was −29.3 mV, which demonstrated that LP-pHS-T2 was stable at room temperature (40). The PDI value was 0.216, which indicated a moderate dispersion of nanoparticles (data not shown). The morphology of LP-pHS-T2 was found to be homogeneous and spherical when visualized under a TEM (Fig. 2C).

Release profile of LP-pHS-T2 at different time points and pH values

The release profile of T-2 toxin from LP-pHS-T2 in vitro was monitored within 72 h at 37°C (pH 7.4). The results are shown in Fig. 3A. The release profile demonstrated a two-phase downward trend, with fast leakage of T-2 toxin in the first 6 h (~20% released), followed by sustained release up to 48 h (~46% released). From 48 to 72 h, the leakage rate increased (~76% released), until reaching a minimum at 72 h. The stability of LP-pHS-T2 at different pH values is shown in Fig. 3B. The release amount of T-2 toxin was up to 91.2% when the pH was 6.5, which indicated that LP-pHS-T2 may be structurally unstable under this faintly acid condition and release T-2 toxin.

Antitumor activity of LP-pHS-T2
LP-pHS-T2 possesses cytotoxicity effects on tumor cells

MTT assay was employed to test the inhibition rate of T-2 toxin and LP-pHS-T2 on a series of tumor cells and a normal cell line, L929. The IC50 values of each group are shown in Table II. T-2 toxin and LP-pHS-T2 exhibited good antitumor activity at the same concentration (P>0.05), which indicated that the proposed liposomal formulation did not noticeably reduce the therapeutic index and that T-2 toxin could be released gradually. However, possibly due to the sustained release of liposomes, only the IC50 values of LP-pHS-T2 on K562 cells were slightly higher compared with T-2 toxin alone. In addition, the IC50 value of LP-pHS-T2 in normal cells (L929 cells) was significantly higher compared with that of T-2 toxin (P<0.05), which demonstrated the reduction of T-2 side effects as result of its encapsulation.

Table II.

IC50 values of T-2 toxin and LP-pHS-T2 by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay at pH 7.4.

Table II.

IC50 values of T-2 toxin and LP-pHS-T2 by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay at pH 7.4.

Cell lineT-2 toxin, ng/mlLP-pHS-T2, ng/ml
A549150.68±0.85174.38±2.46
HepG-2210.41±8.14253.41±5.47
MKN-45213.13±6.48249.36±9.61
K56211.59±1.05 43.42±2.87a
L9291437.53±20.80 1864.24±20.47b

a P<0.01

b P<0.05 vs. T-2 toxin group. LP-pHS-T2, liposomal delivery system containing T-2 toxin.

Apoptosis analysis using Hoechst staining

The nucleus of apoptotic cells can be stained dense blue using Hoechst 33342. The results from the present study are shown in Fig. 4A and they demonstrate that T-2 toxin and LP-pHS-T2 at dose of 10 µg/ml markedly induced apoptosis in Hep-G2 cells, as indicated by the enhanced intensity of blue fluorescence.

Wound healing assay

Certain tumor cells are capable of migration. Thus, a wound-healing assay was performed to observe the inhibitory effect of T-2 toxin and LP-pHS-T2 on the migration ability in Hep-G2 cells. The results are shown in Fig. 4B. After a 24-h incubation, the wound areas in the control group were almost healed. By contrast, the migration ability in the Hep-G2 cells was significantly inhibited after T-2 toxin or LP- pHS-T2 treatment at 10 µg/ml as shown in Fig. 5 (P<0.05).

Apoptosis detection via flow cytometry

The nucleic acid dye PI and Annexin V can differentiate early apoptosis from late apoptosis and necrotic cells. As shown in Fig. 6, the percentage of total apoptotic cells increased nearly two-fold after T-2 toxin or LP-pHS-T2 treatment in Hep-G2 cells compared with that in the control group (P<0.05).

Discussion

It is known that T-2 toxin has strong toxicity (41,42) and thus a great advantage in killing cancer cells. However, due to the toxic side effects on normal cells, the use of T-2 toxin in the clinic is limited. Studies on targeted agents appear to be the only solution. Attempts have been made to prepare T-2 toxin-conjugated antibody drugs, but no substantial progress has occurred (43).

As a novel type of nano drug carrier, pH-sensitive liposomes have been widely studied in tumor therapy for their advantages of tumor-targeting and sustained release (44). Changes in pH in the tumor microenvironment can cleave the linkages between liposomes and drugs, and prompt drug release to the specific tumor tissues (45). In a previous study, DOX loaded into pH-sensitive micelles exhibited an enhanced cytotoxic effect in MCF-7 cancer cells (46). Dextran sulfate-DOX and alginate-cisplatin polymer-drug complex-loaded liposomes also exhibit specific receptor-mediated endocytic uptake in cancer cells (47). In the present study, a pH-sensitive liposome containing T-2 toxin was prepared. In the preparation process, the EE value was affected mainly by the type of biomaterials, the loading method, the hydration volume and the drug-lipid ratio. The density of T-2 toxin is twice that of phospholipids. As a fat-soluble small molecule drug, T-2 toxin is mainly wrapped in the voids of the phospholipid bilayer (48,49). In the present study, the packaging of T-2 toxin was achieved under the condition of a higher drug-to-lipid ratio compared with other reports (25,26). Ultrasound and extrusion steps showed significant effects on the particle size. Following ultrasound, the particle size was distributed in a wide range from 100–500 nm, which was improved after extrusion. Following the optimization of the preparation process, the EE value of LP-pHS-T2 reached 95±2.43%. The morphology of LP-pHS-T2 was a spherical shape ~100 nm in diameter. LP-pHS-T2 was stable at pH 7.4. The release profile showed a two-phase downward trend starting with a quick release within 10 h, followed by a slow release until reaching a minimum EE value at 72 h. In the measurement of sensitivity to pH of LP-pHS-T2, the remaining T-2 toxin in the sample was immediately detected after incubation for 30 min. It was identified that LP-pHS-T2 released the most toxin at pH 6.5 in the first 30 min of incubation, which indicated that LP-pHS-T2 was extremely sensitive to this pH value, which may be due to the fusogenic properties of lipids. When the pH decreased to 6.5, the carboxyl groups of DPPC and DOPE were sensitively protonated and formed a hexagonal phase structure, which accelerated the drug release by membrane fusion (50). By contrast, at pH values higher or lower than 6.5, only slight amounts of T-2 toxin were released within 30 min. Based on the natural active targeting properties of liposomes, it can be reasonably hypothesized that LP-pHS-T2 may target the pathological tissues, including cancer, inflammation and infection sites, and ischemic areas, in which the pH is known to be lower compared with normal tissue (51).

The antitumor effects of LP-pHS-T2 were tested on a series of tumor cells in vitro by MTT assays, with T-2 toxin as the control. The data demonstrated that LP-pHS-T2 can inhibit the proliferation of carcinoma cells. Different types of cells exhibited different degrees of tolerance; K562 cells were most sensitive to T-2 toxin compared with A549, Hep-G2, MKN-45 and L929 cell lines. The difference in IC50 values for different cancer cells was due to the toxic mechanism of T-2 toxin. T-2 toxin inhibits cell proliferation by inhibiting some key enzymes involved in protein and nucleic acid synthesis (8). So the greater toxicity of T-2 toxin might be observed in the more vigorously proliferative cells. Furthermore, the IC50 value of LP-pHS-T2 on L929 cells was increased by up to 1.3-fold compared with that of T-2 toxin, which indicated that the side effects of T-2 toxin were decreased. However, although these IC50 values were statistically different, as a potential antitumor drug, the safety of LP-pHS-T2 has not been able to meet clinical requirements due to the lack of preclinical studies and its toxicity to normal cells. It remains necessary to modify or further verify its safety through more experiments. Following LP-pHS-T2 and T-2 toxin treatment, apoptosis and cell death occurred, and the migration ability of Hep-G2 cells was significantly inhibited. However, compared to the non-treated group, T-2 toxin and LP-pHS-T2 only caused a slightly increased cell cycle arrest at the G0/G1 phase. The mechanism of apoptosis induced by T-2 toxin may be a non-cell cycle dependent pathway (data not shown). Moreover, considering that the pH-sensitive liposomes can be recognized and sequestered by the phagocytes of the reticulo-endothelial system (RES), the clinical use of LP-pHS-T2 remains a future prospect. To avoid their uptake by RES, further reduce the side effects and prolong circulation time, grafting of the liposomal membranes with pegylated phospholipids (52), construction of a programmed nano-selenium overcoat nanoparticles for T-2 toxin (53), or combination of T-2 toxin and multiple targeting carriers may be the solution for LP-pHS-T2 targeted therapy (54).

In summary, the present study investigated LP-pHS-T2, a novel pH-sensitive liposome delivery system containing T-2 toxin; it not only has a release ability in the tumor microenvironment, but also has advantageous antitumor activity in vitro. Additionally, due to the encapsulation of liposomes, the side effects of T-2 toxin are relatively reduced. The present study provided a novel approach for the development of T-2 toxin-based anticancer drugs. However, it is important to note that the mechanism and the modification of LP-pHS-T2 on tumor cells require further studies.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

This study was supported by the Program of Administration of Traditional Chinese Medicine of Jilin Province, P.R. China (grant no. 2020132), the Research and Development of Industrial Technology' Program of Jilin Province, P.R. China (grant nos. 20180623045TC and 20170204005YY), the Program of Jilin Science and Technology Bureau, P.R. China (grant nos. 2019001179 and 20200104093) and the Start Funding of Jilin Medical University (grant no. 2017kyqd001).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YD and YL prepared the liposomes. GM performed the HPLC. JG was responsible for cell culture. MY evaluated the pH sensitivity and stability of liposomes. HX analyzed the data regarding the characterization of liposomes. JZ and WZ investigated the antitumor activity of drugs. ML and YL optimized the preparation conditions of liposomes. HW analyzed all the data and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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November-2020
Volume 22 Issue 5

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Spandidos Publications style
Dong Y, Meng G, Guo J, Yin M, Xu H, Li Y, Zhu J, Zhu W, Li M, Li Y, Li Y, et al: Preparation of T‑2 toxin‑containing pH‑sensitive liposome and its antitumor activity. Mol Med Rep 22: 4423-4431, 2020
APA
Dong, Y., Meng, G., Guo, J., Yin, M., Xu, H., Li, Y. ... Wang, H. (2020). Preparation of T‑2 toxin‑containing pH‑sensitive liposome and its antitumor activity. Molecular Medicine Reports, 22, 4423-4431. https://doi.org/10.3892/mmr.2020.11531
MLA
Dong, Y., Meng, G., Guo, J., Yin, M., Xu, H., Li, Y., Zhu, J., Zhu, W., Li, M., Li, Y., Wang, H."Preparation of T‑2 toxin‑containing pH‑sensitive liposome and its antitumor activity". Molecular Medicine Reports 22.5 (2020): 4423-4431.
Chicago
Dong, Y., Meng, G., Guo, J., Yin, M., Xu, H., Li, Y., Zhu, J., Zhu, W., Li, M., Li, Y., Wang, H."Preparation of T‑2 toxin‑containing pH‑sensitive liposome and its antitumor activity". Molecular Medicine Reports 22, no. 5 (2020): 4423-4431. https://doi.org/10.3892/mmr.2020.11531