Thermo-sensitive injectable hydrogel loading with elemene-loaded liposomes for enhanced anti-tumor effect

Abstract

Due to the heterogeneity and the complexity of the tumor microenvironment, combination therapy, especially the combination of chemotherapy and photothermal therapy (PTT), had received increasing attention. However, the co-delivery of small molecule drugs for chemotherapy and photothermal agents was a key issue. Herein, we prepared a novel thermo-sensitive hydrogel loading with elemene (ELE)-loaded and nano graphene oxide (NGO)-based liposomes for enhanced combined therapy. ELE was applied as the model drug for chemotherapy because it was a natural sesquiterpene drug with broad-spectrum and efficient antitumor activity. NGO was applied as drug carrier and photothermal agent simultaneously due to its two-dimensional structure and high photo-thermal conversion efficacy. NGO was further modified with glycyrrhetinic acid (GA) to improve its water dispersion, biocompatibility and tumor-targeting ability. ELE was loaded by GA-modified NGO (GA/NGO) to prepare the liposomes designated as ELE-GA/NGO-Lip, which was further mixed with chitosan (CS) solution and β-glycerin sodium phosphate (β-GP) solution to prepare the thermo-sensitive hydrogel designated as ELE-GA/NGO-Lip-gel. The obtained ELE-GA/NGO-Lip-gel had the gelling temperature of 37°C, temperature and pH-response gel dissolution and high photo-thermal conversion effect. More importantly, ELE-GA/NGO-Lip-gel upon 808 nm laser irradiation had relative high anti-tumor efficiency against SMMC-7721 cells in vitro. This research might provide a potent platform for the application of thermos-sensitive injectable hydrogel in combined tumor therapy.

Keywords

Combined therapy thermo-sensitive hydrogel liposomes elemene nano graphene oxide glycyrrhetinic acid

Introduction

Malignant tumor has become one of the major public health problems in the world. For nearly half a century, malignant tumor has seriously threatened human life and health as a major disease in clinical medicine.¹ The traditional treatment methods of malignant tumors mainly include chemical therapy, radiotherapy and surgical treatment, which are easy to affect normal tissues and cells, and then lead to serious toxic and side effects.^2–4 Elemene (ELE) is a natural sesquiterpene anticancer drug with anticancer activity extracted from Curcuma vulgaris for the first time in China, whose chemical name is 1-methyl-1-vinyl-2, 4-diisopropyl cyclohexane.⁵ The results of its clinical pharmacological studies show that ELE has the outstanding advantages of broad anti-tumor spectrum, definite curative effect, mild toxic and side effects, and no myelosuppression.⁶ At present, ELE alone or in combination with other chemotherapy drugs has achieved remarkable efficacy in the treatment of liver cancer, breast cancer and digestive tract tumors. The antitumor mechanism of ELE mainly includes inducing cell apoptosis, inhibiting tumor cell migration and invasion, inhibiting tumor angiogenesis, reversing drug resistance and sensitization, and inducing protective autophagy.^7,8 ELE can also improve the immune function of the host, enhance the effectiveness of chemotherapy, and reduce toxic secondary reactions.⁹ However, since ELE has very little polarity and is insoluble in water and it cannot be absorbed by the body, which hinders its wide clinical application. In addition, ELE intravenous injection is prone to inflammation, allergy and other adverse reactions, resulting in its clinical use is limited.¹⁰ Therefore, in order to improve the biological properties and bioavailability and reduce adverse reactions of ELE, scientists have been working on the development of novel delivery systems for ELE.

Liposomes is a new nano drug delivery system that has been actively studied in the field of pharmacy in recent years. It has the advantages of good histocompatibility, cell affinity, targeting and sustained release, and is widely used to load anti-tumor drugs to improve the bioavailability of insoluble drugs and reduce adverse reactions.¹¹ Thermo-sensitive hydrogels form a three-dimensional network structure through chemical cross-linking or physical interaction of macromolecules in aqueous solution, which is highly porous in the swelling state and can wrap drugs, liposomes, biomolecules, etc.¹² Thermo-sensitive hydrogel for injection is a liquid solution with good fluidity at room temperature and becomes a semi-solid gel state at 37°C in the physiological environment. The drug is dissolved or suspended in the solution that can form hydrogel, which can be used for local tissue and minimally invasive injection. Thermo-sensitive hydrogel can realize the sustained release and long-term effect of the drug in the local tissue, thus improving the drug efficacy and reducing the adverse reactions.^13,14 Relevant studies have shown that the administration of thermo-sensitive hydrogels through intratumoral injection (i.t.) can increase the accumulation of drugs at the tumor site and achieve accurate targeted distribution.^15,16 At present, liposome-loaded hydrogel system has been widely used. Liposomes containing drugs are embedded in hydrogel polymer matrix to prepare liposome-loaded gel delivery system.¹⁷ When liposomes act as drug reservoirs, hydrogels provide additional diffusion resistance, which can prolong drug release, avoid sudden release effects, increase local drug delivery concentration, reduce side effects, and improve drug efficacy.^18,19

In order to improve the therapeutic effect of cancer, light therapy (mainly including photo-thermal therapy and photodynamic therapy) has attracted more and more attention because of its good tissue penetration and less toxic side effects. Photothermal therapy (PTT) is a new minimally invasive treatment for cancer, which is a non-invasive, safe and mild treatment strategy compared with chemotherapy.²⁰ The principle of PTT is that photothermal transduction agent with high efficiency is injected into the body and photosensitizer is targeted accumulated in the tumor site with drugs. Light energy is converted into heat energy by irradiation with lasers such as near-infrared transduction. Killing tumor cells without damaging normal tissue through PTT is achieved by making the temperature of tumor or cancerous tissue rise to the range of effective treatment temperature and making use of the difference of temperature tolerance between tumor tissue and normal tissue.^21–23 Nano graphene oxide (NGO) is a new type of two-dimensional carbon nanomaterials with excellent electrical, optical, mechanical and thermal properties, and is a new photosensitizer material.^24,25 NGO has a high absorption of near-infrared light and high photo-thermal conversion efficiency. It can be applied to the preparation of photo-responsive nanocomposite hydrogels with a large number of oxygen-containing functional groups (hydroxyl, carboxyl and epoxy groups) on the surface, which greatly improves the hydrophilicity and biocompatibility of NGO.^26,27 The anti-tumor mechanism of PTT generally includes inducing tumor cell apoptosis, synergistic effect, inducing immune function activation, overcoming tumor multidrug resistance and so on.²⁸ Due to the tumor site with enhanced permeability and retention (EPR) effect, rich blood vessels, wide hemal wall gap, the integrity in tumor tissues is poor and lack of lymphatic circumfluence, which can make a few small molecular substance and nano liposomes selectively high permeability and retention in tumor site, thus increasing the accumulation of drug in the tumor site to improve tumor therapy.^29–31

In this study, ELE was used as an antitumor model drug, glycyrrhetinic acid (GA)-modified graphene oxide (GA/NGO) was used as a carrier and photosensitizer, chitosan (CS) and β-sodium glycerophosphate (β-GP) were used as the main matrix materials to form hydrogel. The thermo-sensitive hydrogel containing ELE-loaded and GA/NGO-based liposomes was prepared by reverse-phase evaporation method and cold solution method and designed as ELE-GA/NGO-Lip-gel (Figure 1). ELE loading on GA/NGO was possibly through π-π stacking and electrostatic interactions,³² ELE-GA/NGO and free ELE were respectively entrapped into the inner aqueous core and membrane regions of the liposomes, then ELE-GA/NGO-Lip was incorporated into the hydrogel substrates to form ELE-GA/NGO-Lip-gel.^33,34 The experimental results showed that ELE-GA/NGO-Lip-gel had good temperature sensitivity and biodegradability, and could be used for local injection of tumor. Then, the antitumor activity of ELE-GA/NGO-Lip-gel with 808 nm (2.5 W/cm²) NIR laser irradiation was investigated in SMMC-7721 cells in vitro, including cytotoxicity assay, cell migration assay and cell uptake assay, etc.

Materials and methods

Materials

Standard β-elemene (ELE, purity >99.4%) was supplied by National institute for food and drug control (Beijing, China). β-Elemene (ELE, purity >95.0%) was purchased from Suzhou NMT Biotech Co, Ltd (Suzhou, China). Graphene oxide (NGO, 150 nm, purity >95%) was purchased from Nanjing Jicang Nano Technology Co Ltd (Nanjing, China). Glycyrrhetinic acid (GA, purity >98%) was bought from Wuhan Yuancheng Co-Creation Technology Co, Ltd Soy lecithin was purchased from Tianjin Guangfu Fine Chemical Research Institute. Cholesterol was purchased from Chuangsheng Biotechnology Co, Ltd CS (η ∼ 100 mPs) was purchased from Shanghai Ruiyong Biotech Co, Ltd β-Glycerin sodium phosphate (β-GP) was bought from Shanghai Aladdin Co, Ltd (Suzhou, China). RPMI 1640 medium and phosphate buffer (PBS) were purchased from Beijing Solarbio Science and Technology Co, Ltd Fetal bovine serum (FBS) was supplied by Zhejiang Tianhang Biotechnology Co, Ltd Dimethyl sulfoxide (DMSO) were purchased from Beijing Dingguo Changsheng Biotechnology Co, Ltd IR783 dye was purchased from TCI (Shanghai) Chemical Industry Development Co, Ltd All materials were of analytical grade. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Fluorescein isothiocyanate (FITC) were purchased from Beijing Aotuoda Science and Technology Co, Ltd Dihydrochloride Staining Solution (DAPI) was Bought Shanghai Beyotime Biotech Co, Ltd SMMC-7721 human liver cancer cells were friendly provided from Prof. Zhang of Zhengzhou University.

Preparation of ELE-GA/NGO-Lip-gel

ELE-GA/NGO-Lip was prepared by reverse phase evaporation method.³⁵ Soy lecithin and cholesterol were dissolved in 20 ml of chloroform and the mixture was placed into eggplant-shaped bottle for rotary evaporation at 40°C. After added 5 ml of ELE-GA/NGO solution containing 5.0 mg ELE and 90 mg of GA/NGO, the mixture was water-bath sonication for 6 min. Remove the organic solvent under reduced pressure at 40°C for 30 min by using a rotary evaporation apparatus. The initial liposomes emulsion was further ultrasonicated in ice bath by an ultrasonic cell disruptor (80 W, 3 min) and ELE-GA/NGO-Lip was obtained. The blank liposomes (GA/NGO-Lip) was prepared by using the same method.

ELE-GA/NGO-Lip-gel was performed as following.³⁶ First, 2.5 g of CS was dissolved in 100 ml of 0.1 mol/L HCl and was stirred at 100 rpm until the solution was clear (solution A), and 0.8 g of β-glycerophosphate (β-GP) was dissolved in 1 ml of ultrapure water (solution B). We then added solution B into solution A slowly under ice-bath-stirring at 100 rpm for 10 min, then CS/β-GP hydrogel was obtained. ELE-GA/NGO-Lip was dropped into the CS/β-GP gel (1:2), the mixture was stirred at room temperature for 10 min and then ELE-GA/NGO-Lip-gel was obtained.

Characterization of ELE-GA/NGO-Lip-gel

The microstructure of ELE-GA/NGO-Lip was observed by TEM (JEM-1400, JEOL, Japan) at an acceleration voltage of 100 kV. ELE-GA/NGO-Lip was diluted in PBS and placed on carbon-coated copper grids with 3% (w/v) phosphotungstic acid staining for 3 min before further analysis. The particle size and zeta potential of ELE-GA/NGO-Lip were measured using a DLS (Zetasizer Nano ZS-90, Malvern, UK). Drug encapsulation efficiency (EE %) was determined by using a combined centrifugation with an ultra-filtration method. The concentration of ELE was determined by high performance liquid chromatography (HPLC, Waters e2695, Waters, USA). Methanol: water (95:5, v/v) was applied as the mobile phases, and ran at a flow rate of 1.0 ml/min. The column effluent was monitored with a UV detector at λ = 207 nm.

The morphology of ELE-GA/NGO-Lip-gel was observed with scanning electron microscope (SEM, QVANTA FEG-400, FEI, USA). The gelation time and gelation temperature of ELE-GA/NGO-Lip-gel were measured by the vial inverting method with the temperature rises rate at 0.1°C each time. And the rheological measurement of ELE-GA/NGO-Lip-gel was determined by using a rotary viscosimeter (NDJ-5S, Fangrui, Shanghai, China)³⁷. The degradation tests in vitro of ELE-GA/NGO-Lip-gel at 37°C and 42°C were also inspected at with phosphate buffer (PBS, pH 5.0 and pH7.2) as the degradation liquid. The stability of ELE-GA/NGO-Lip-gel in various media were investigated by incubating them in purified water, PBS (pH 7.4), normal saline (NS), complete medium, FBS at room temperature for up to 7 days. The appearance of precipitation was observed by visual inspection.

Photothermal conversion efficiency

For the in vitro laser radiation experiments, an 808 nm laser emitter with a power density of 2.5 W/cm² (Changchun laser station, Changchun, Chinese) was chosen as the NIR light source. ELE-GA/NGO-Lip-gel at 0, 0.1, 0.2 and 0.5 mg/ml was exposed to NIR laser irradiation, and the temperature was measured and recorded. To display the photo-thermal conversion features more intuitively, an infrared thermal imager (Ti-200, FLUKE, USA) was used to collect the infrared thermal images of ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel after irradiating with 808 nm (2.5 W/cm²) laser for 0, 1, 2 and 3 min, with purified water as the control.

Antitumor activity of ELE-GA/NGO-Lip-gel in vitro

Cell viability

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to evaluate the in vitro cytotoxicity of the prepared liposomes³⁸. SMMC-7721 cells were seeded into a 96-well plate at a density of 2 × 10⁵ cells per well and incubated at 37°C in a humidified 5% CO₂ atmosphere. After 24 h, the cells were treated with GA/NGO-Lip-gel at different GA/NGO concentrations from 10 to 100 μg/ml or ELE-loaded preparations including ELE, ELE-GA/NGO-Lip or ELE-GA/NGO-Lip-gel at different ELE concentrations from 10 to 100 μg/ml). The blank control group was complete culture medium, and the negative control group was cell suspension (no drug solution or blank carrier). The 96-well plates were cultured in an incubator for 24, 48 and 72 h, respectively. The 96-well plates were taken out and 20 μl MTT solution (5 mg/ml) was added to each well, then the plates were incubated for another 4 h in the dark. The upper culture medium was discarded and 150 μl DMSO was added in each well to dissolve the formazan crystals, followed by shaking at room temperature for 10 min. The optical density (OD) value was measured by a microplate spectrophotometer (Bio Rad, USA) at 490 nm. The cell survival rate and cell inhibition rate were calculated respectively according to the following formulas. The cell survival rate = [(OD _Sample − OD _Blank)/(A _Negative − A _Blank)] × 100%; the cell inhibition rate = [(A _Negative − A _Sample)/(A _Negative − A _Blank)] × 100%.

In ELE-GA/NGO-Lip + NIR or ELE-GA/NGO-Lip-gel + NIR group, SMMC-7721 cells were treated with 808 nm laser irradiation at a density of 2.5 W/cm² for 2 min immediately after treatment with ELE-loaded preparations. Then the cells were cultured in an incubator for 24 h, other steps were the same as above.

Cell migration assay

Cell migration was evaluated by using a scratch assay. In brief, SMMC-7721 cells were seeded in a 6-well plate (2 × 10⁵) and a pipette was used to create a straight scratch, three straight scratches for each well (n = 3). After that the cells were treated with ELE sol., ELE-GA/NGO-Lip or ELE-GA/NGO-Lip-gel at ELE concentrations of 20 μg/ml. The blank control group was added complete culture medium. The cells contained ELE-GA/NGO-Lip + NIR or ELE-GA/NGO-Lip -gel + NIR were treated with 808 nm laser irradiation at a density of 2.5 W/cm² for 2 min after treatment with ELE-loaded preparations. After 24-hour incubation, the scratch healing was captured using Olympus inverted microscope (Olympus, Tokyo, Japan) and the distance from and side of the scratch to the other was measured. Scratches areas were calculated was measured by using Image J software. The cell migration rate = [(Scratch area _0h − Scratch area _24h)/(Scratch area _0h)] × 100%.

Cell uptake assay

Flow cytometry analysis was used to evaluate the intracellular uptake capacity of ELE-GA/NGO-Lip-gel in SMMC-7721 cells. FITC, a fluorescence probe, was incorporated into GA/NGO-Lip as well as GA/NGO-Lip-gel by mixing of these samples with FITC according to the following method: FITC solution (20 μg/ml) was added and ultra-sonicated with ultrasonic cell disruption system to obtain GA/NGO-Lip-FITC and GA/NGO-Lip-gel-FITC. SMMC-7721 cells were seeded in 12-well plates with a density of 1 × 10⁴ cells per well. When cells reached 80% ∼ 90% confluence, they were treated with GA/NGO-Lip-FITC and GA/NGO-Lip-gel-FITC at 37°C for 1 h, 2 h and 4 h, respectively. At the designated time points, the cells were washed three times with sterile PBS for three times. After added 500 μl of PBS, the cells were observed under fluorescence microscopy (OLYMPUS IX73, Japan) and the images were recorded, and the results were analyzed with the software Image Proplus. In order to further research the cellular uptake quantitatively, the collected cells were measured by using a flow cytometer (Partech GmbH, Germany).

SMMC-7721 cells seeded in confocal special small dishes (Φ = 35 mm) with a density of 2 × 10⁴ cells per dish. When cells reached 80% confluence, they were treated with FITC, GA/NGO-Lip-FITC or GA/NGO-Lip-gel-FITC (the concentration of FITC: 20 μg/ml) at 37°C for 2 h. At the designated time points, the cells were washed three times with sterile PBS followed and then stained with 200 μl DAPI (100 μg/ml) for 15 min under a dark incubating condition. The cells were washed three times with PBS to remove the dye. After added 500 μl of PBS the cell uptake images were obtained by using a confocal laser scanning microscope (FV1200, OLYMPUS, Japan).

Statistical analysis

All values were expressed as mean ± standard deviation (SD). All experiments were repeated at least three times with a minimum sample size of three. Comparisons having p value <0.05 were considered significant.

Results and discussion

Preparation and characterization of ELE-GA/NGO-Lip-gel

The physical and chemical properties of ELE-GA/NGO-Lip-gel are shown in Figure 2. As shown in Figure 2(a), the blank liposomes, ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel had different colors but they are all relatively homogeneous. The blank liposomes and ELE-GA/NGO-Lip both had almost spherical shape (Figure 2(b) and (c)), both blank hydrogel and ELE-GA/NGO-Lip-gel samples exhibited microporous structure (Figure 2(e) and (f)). The result of HPLC determination showed the concentration of ELE in ELE-GA/NGO-Lip was 9.0 mg/ml and the EE and DL were as high as 99.01 ± 0.57% and 13.70 ± 0.49%, respectively. After being left at room temperature for 7 days, a homogeneous solution without obvious precipitation could be obtained, indicating that ELE-GA/NGO-gel had good stability in various mediums (Figure 2(d)). The viscosity of ELE-GA/NGO-Lip-gel was constant when the temperature was about 25°C and began to surge at 30°C and increased slowly at 37°C (Figure 2(g)).³⁴ The particle sizes of ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel were 141.7 ± 0.75 nm (PDI = 0.13, Figure 2(h)) and 171.0 ± 0.25 nm (PDI = 0.13) (Figure 2(i)), respectively. Zeta potential of ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel were −25.00 ± 0.23 mV and 8.06 ± 4.9 mV, respectively.

The infrared thermal imaging results of ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel with 808 nm of laser irradiation were shown in Figure 3(a). The figure showed that after irradiation for 1 min, the temperatures of ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel rose to 36.7°C and 41.0°C, respectively. After irradiation for 3 min the temperatures were 48.0°C and 51.2°C, respectively, while the temperature of the water just increased from 26.0°C to 28.6°C at the same condition. Figure 3(b) showed the photo-thermal conversion curves of ELE-GA/NGO-Lip-gel in different concentrations after irradiation for 10 min. In accordance with the results in Figure 3(a), the photo-thermal conversion curves of ELE-GA/NGO-Lip-gel presented the concentration and time-dependence.

The degradation curves of ELE-GA/NGO-Lip-gel at 37°C and 42°C were demonstrated in Figure 3(c) and (d). As seen from Figure 3(c) and (d), ELE-GA/NGO-Lip-gel had the characteristics of slow dissolution under different conditions, and demonstrated temperature and pH-responsive. At 37°C and pH 5.0, ELE-GA/NGO-Lip-gel dissolved rapidly and the cumulative dissolution rate was 68.01 ± 3.48% in the first 3 h, and then slowly to obtain the almost complete dissolution with the cumulative dissolution rate of 90.52 ± 3.27% in 7 h and the cumulative dissolution rate was 90.52 ± 3.27%. At 42°C and pH 5.0, the change range of gel dissolution rate first increased and then slowed down. The cumulative dissolution rate was 77.89 ± 3.27% at 3 h and 95.26 ± 3.3% at 7 h. According to the fitting results of the mathematical model of the dissolution curve, the dissolution process of ELE-GA/NGO-Lip-gel in different environments in vitro had a good fit to the first-order kinetic equation (Table 1), indicating that ELE-GA/NGO-Lip-gel had a sustained release effect. The results could lay a theoretical foundation for the following combination of PTT and chemotherapy.

Table 1.

Mathematical model fitting of ELE-GA/NGO-Lip-gel dissolution curves under different conditions.

	Model	Equation	R ²
37°C, pH 7.2	Zero-order equation	y = −4.76x + 11.87	0.93473
	First-order equation	y = −58.34 (1 − e^0.13x)	0.96636
	Higuchi equation	y = 42.51x^1/2 − 39.14	0.86623
37°C, pH 5.0	Zero-order equation	y = 27.22x + 10.07	0.83867
	First-order equation	y = −98.54 (1 − e0.35x)	0.97857
	Higuchi equation	y = 38.78x^1/2 − 7.15	0.92768
42°C, pH 7.2	Zero-order equation	y = 10.84x + 10.26	0.92095
	First-order equation	y = 120.73 (1 − e^0.15x)	0.93474
	Higuchi equation	y = 38.22x^1/2 − 21.73	0.94045
42°C, pH 5.0	Zero-order equation	y = 25.59x + 11.42	0.66158
	First-order equation	y = 105.36 (1 − e^0.35x)	0.87365
	Higuchi equation	y = 45.24x^1/2 − 15.84	0.79082

Antitumor activity in vitro of ELE-GA/NGO-Lip-gel

Cell viability

The effects of different concentrations of GA/NGO-Lip-gel on the cell growth of SMMC-7721 after 24 h and 48 h incubation were shown in Figure 4(a). As can be seen from the figure, the survival rate of SMMC-7721 cells decreased slightly with the increase of concentration and the extension of incubation time. The survival rates of SMMC-7721 cells were 103.04% ∼ 113.70% at 24 h, and 82.71%∼106.25% at 48 h, indicating that GA/NGO-Lip-gel had no significant effect on the proliferation of SMMC-7721 cells.³⁹

The cell inhibition rates of SMMC-7721 treated with ELE sol., ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel at different concentrations and incubation time were shown in Figure 4(c)–(e). With the increase of concentration and the prolongation of incubation time, the cell inhibition rate of each group increased gradually. After incubation for 72 h, the cell inhibition rates of ELE sol., ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel (ELE 100 μg/ml) were 62.59 ± 3.30%, 84.66 ± 1.48% and 96.04 ± 1.45%, respectively. The results showed that ELE-GA/NGO-Lip-gel had high toxicity to SMMC-7721 cells, and could effectively inhibit the proliferation of SMMC-7721 cells in a concentration and time-dependent manner.

SMMC-7721 cells treated with ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel after 24 h were also irradiated with 808 nm of laser. It could be seen from Figure 4(b), the inhibition rate of SMMC-7721 cells in ELE-GA/NGO-Lip group and ELE-GA/NGO-Lip-gel group increased obviously when the concentrations were 10, 20, 50 and 100 μg/ml respectively. The inhibition rate of SMMC-7721 cells increased from 75.31 ± 1.99% (without laser irradiation) to 82.14 ± 1.89% (with laser irradiation) at ELE concentration of 100 μg/ml). The effects of PTT were significant, and the differences in other concentrations were statistically significant.

Cell migration assay

The migration abilities of SMMC-7721 treated with ELE sol., ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel for 24 h were shown in Figure 5. As seen from Figure 5(a) and (b), the migration abilities of SMMC-7721 cells in ELE sol., ELE-GA/NGO-Lip and ELE-GA/NGO-Lip-gel groups were all significantly inhibited.⁴⁰ And the migration rates of SMMC-7721 cells in blank group, ELE-GA/NGO-Lip group and ELE-GA/NGO-Lip-gel group were 31.90 ± 2.71%, 107.36 ± 2.85% and 115.03 ± 3.67%, respectively. The migration inhibition rate of SMMC-7721 cells in ELE-GA/NGO-Lip-gel + NIR group was 118.28 ± 2.75%. The results showed that ELE-GA/NGO-Lip-gel had a significant inhibitory effect on the migration of SMMC-7721 cells, and the effect when combined with NIR laser irradiation was more significant (p < 0.001).

Cell uptake assay

The uptake effects of SMMC-7721 cells on FITC, GA/NGO-Lip-FITC and GA/NGO-Lip-gel-FITC were investigated by the inverted fluorescence microscopy.^36,41 As seen from Figure 6(a), the fluorescence distribution of each group was uniform and the cell density was moderate. The fluorescence intensity of FITC group reached the highest at 2 h, and then decreased gradually. These results indicated that the uptake of the drug solution by SMMC-7721 cells was limited and quickly metabolized. At 1 h, 2 h and 4 h, the fluorescence signals of GA/NGO-Lip-FITC and GA/NGO-Lip-gel-FITC groups were stronger with time-dependence, and the fluorescence intensity was stronger than that of FITC group. The fluorescence intensity of GA/NGO-Lip-FITC group reached the peak value at 2 ∼ 4 h, which was higher than that of GA/NGO-Lip-gel-FITC GA/NGO-gel group. The software ImageProPlus was used to perform semi-quantitative analysis on the fluorescence intensity in the images, and Figure 6(b) was obtained. As seen from Figure 6(c), the fluorescence intensity in SMMC-7721 cells from strong to weak was in the order of GA/NGO-Lip, GA/NGO-Lip-gel, and FITC. The result showed that GA/NGO-Lip and GA/NGO-Lip-gel could effectively promote the uptake of ELE by SMMC-7721 cells and they both had sustained release properties.

Conclusion

In this study, a thermo-sensitive hydrogel ELE-GA/NGO-Lip-gel containing ELE-loaded and GA/NGO-based liposomes (ELE-GA/NGO-Lip-gel) was successfully constructed for the combination of PTT and chemotherapy. The gelling temperature of ELE-GA/NGO-Lip-gel was suitable, the encapsulation rate and drug loading rate were high, and the intelligent hydrogel had good power and time-dependent photothermal conversion characteristics. Studies on anti-tumor activity in vitro showed that ELE-GA/NGO-Lip-gel had good anti-tumor activity especially when combining with 808 nm laser irradiation, which may provide certain research ideas for the application of thermo-sensitive hydrogel in tumor therapy.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grant from the National Natural Science Foundation of China (No. 81803740 and No. 82003298), Key Scientific Research Project of Colleges and Universities in Henan Province (No. 23A360007).

ORCID iD

Xiali Zhu

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