Lung cancer with high morbidity is usually accompanied by distant metastases in most clinically diagnosed cases,1 which results in higher mortality than other cancer types.2 Currently, platinum drugs in combination with other medications are usually adopted for the treatment of late period lung cancer.3 The cisplatin (DDP) forms platinum‐DNA adduct with DNA upon entering cells to interfere with the DNA replication and transcription, leading to DNA mismatch and inducing cell apoptosis.4 Free DDP is prone to cause multidrug resistance (MDR) and many adverse effects. Therefore, it has become a research hotspot to find drugs with high efficiency and low toxicity that can enhance the sensitivity of chemotherapy.5
As a phytochemical component, oleanolic acid (OA) is widely found in plants such as Panax japonicus, hawthorn, clove, jujube, loquat leaf, and so on. OA is of low toxicity and has a variety of biological activities, such as antivirus, immune enhancement and immunosuppression (two‐way regulation of immunity), hypolipidemic and liver protection effects. In particular, OA is of great significance to the recovery of immunologic function caused by chemotherapy of malignant tumors.6,7
The combined use of chemotherapy drugs can produce antagonistic or synergistic effects. For the synergistic effect, due to the different mechanisms of chemotherapy drugs, MDR can be prevented to a certain extent.8,9 Nanocarriers can increase the solubility of drugs, prolong the blood circulation time of drugs, deliver the drugs to the target tumor sites and release drugs in an environment‐responsive way, contributing significantly to enhance the therapeutic effect and reduce unwanted adverse effects.10,11
In recent years, mesoporous silica nanoparticles (Nsi) have attracted more and more attention because of their low toxicity and high efficiency.12 Compared with traditional nanodrug carriers, such as liposomes and polymer nanoparticles, Nsi has obvious advantages: (a) Highly‐ordered channel structure, which can effectively carry various substances; (b) High specific surface area and large pore volume, which can realize decent drug‐loading; (c) The size distribution of Nsi are controllable through simple regulation of synthesis factors to satisfy different needs. (d) More importantly, the high‐loading and co‐loading capacity of Nsi makes it possible to overcome multiple drug resistance in tumors.13 Some studies14–16 have explored the combination therapy in lung cancer using Nsi as the carrier. Munaweera et al14 reported the nitric oxide‐ and DDP‐releasing Nsi against non‐small cell lung cancer, which successfully restored the sensitivity of resistant populations to the toxicity of chemotherapeutics. Dilnawaz et al15 observed a comparative therapeutic efficacy in lung cancer via the co‐delivery of carfilzomib and survivin siRNA. Zhang et al16 applied co‐delivered DDP prodrug with chlorin e6 by Nsi for chemo‐photodynamic combination therapy. Various methods of combination therapy have unique characteristics and also show superior antitumor effects. Herein this article, we established a Nsi‐based drug delivery system (DDS) containing DDP and OA. We hypothesized that facilitated by the targeting effect of Nsi via the enhanced permeability and retention (EPR) effect, the nanocarriers reached the tumor site and released DDP and OA simultaneously. OA had a sensitizing effect on DDP, which could enhance cell apoptosis, reduce cell resistance, and effectively kill lung cancer cells. We investigated the cytotoxic effects of the DDS in vitro and the antitumor effects in vivo.
Cisplatin (DDP, Pt, 65%) was purchased from the Macklin Biochemical Co., Ltd (Shanghai, China). Oleanolic acid (OA) was obtained from the Aladdin Industrial Co. (Shanghai, China), tetraethyl orthosilicate (TEOS), Triton X‐100 and Methyl thiazolyl tetrazolium (MTT) were obtained from the Sigma‐Aldrich (St. Louis, MO). Other chemicals and reagents were of analytical pure.
DDP‐resistant human lung adenocarcinoma cell line (A549/DDP cell) was supplied by the Chinese Academy of Sciences, Shanghai, China.
The fabrication of DDP‐Nsi was conducted referring to the previous report.17 First, a water‐in‐oil microemulsion was prepared by adding Triton X‐100 (3.30 g), cyclohexane (10.00 g), n‐hexanol (2.23 g), (a certain amount of DDP was needed for the fabrication of DDP‐Nsi) and stirred for 30 min at 25°C. Then TEOS (289.60 mg), and NH4OH (155.80 mg) were successively added to trigger the formation of silica matrix and polymerization. After reacting for 24 h, the Nsi (DDP‐Nsi) was precipitated using an excess amount of ethanol and collected using centrifugation (3000 rpm, 10 min).
For the fabrication of OA‐Nsi (or DDP/OA‐Nsi), the OA was dissolved in ethanol and added into the aqueous solution of Nsi (or DDP‐Nsi) at various wt/wt ratios with stirring for 24 h, and was precipitated to obtain DDP/OA‐Nsi.
The particle size and surface potential were measured by a zetasizer (Zetasizer Nano ZS, Malvern, UK). The DDP content in the nanocarriers was determined by high‐performance liquid chromatography (HPLC) (Agilent 1260) using the following conditions: C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase contained methanol and water (1:1, vol/vol), flow rate = 0.7 ml/min, column temperature = 40°C, UV detector at the detection wavelength of 254 nm for DDP. The OA content was detected by the same column, mobile phase was composed of 0.1% trifluoroacetic acid in water and a mixture of acetonitrile: methanol (17:1, vol/vol) at a ratio of 10:90 (vol/vol), and the temperature was set at 30°C, flow rate = 1.3 ml/min, detection wavelength = 210 nm. The drug loading efficiency of nanocarriers was determined using the following equation:[Image Omitted. See PDF]
The stability of the nanoparticles was assessed in phosphate buffer saline (PBS) (pH 7.4) and mouse plasma using the previous reported protocol.18 In brief, the nanoparticles were diluted using the corresponding medium at the volume ratio of 1:10 and then placed at room temperature for 6 days. The changes in the size of nanoparticles were monitored using a zetasizer and plotted against time.
MTT assay was employed to evaluate cell viability when various formulations were added. First, A549/DDP cells were cultured in 96‐well plates at 6 × 103 cells per well for 24 h. Different concentrations of Nsi, OA‐Nsi (concentrations of nanoparticles: 20, 60, 90, 140, 220, 300 μg/ml), DDP, DDP‐Nsi, DDP/OA‐Nsi, OA or OA‐Nsi (DDP concentrations: 1.5, 3, 6, 12, 30, 60 μM for DDP, DDP‐Nsi, DDP/OA‐Nsi, OA concentrations: 1.5, 3, 6, 12, 30, 60 μM for OA, OA‐Nsi) was added for 48 h. Then MTT (5 mg/ml, 20 μl) was added to each well and cultured at 37°C for 4 h. After 4 h, the supernatant was removed, and DMSO (200 μl) was added to dissolve the precipitation for 30 min. Then the absorbance of each well was measured at 570 nm by a microplate reader (Beckman Coulter, Brea, CA).
A549/DDP cells were cultured in 6‐well plates at the density of 1 × 105 cells/well. Then A549/DDP cells were exposed to OA (50 μM), DDP (3 μM), OA‐Nsi (OA concentration = 50 μM), DDP‐Nsi (DDP concentration = 3 μM), DDP/OA‐Nsi (DDP concentration = 3 μM) or without treatment for 24 h, collected and subjected to dispose by Annexin V‐FITC Apoptosis Detection Kit (Solarbio), following the protocol provided by the manufacturer. The samples were analyzed by flow cytometry (ACEA NovoCyte, Agilent, CA).
A549/DDP cells were cultured in 6‐well plates at the density of 1 × 105 cells/well. Then A549/DDP cells were exposed to DDP, DDP‐Nsi, and DDP/OA‐Nsi (DDP concentration: 1.5 μM) for 6, 12, or 24 h, respectively. At the corresponding time point, the cells were collected and treated by radio immunoprecipitation assay (RIPA) lysis buffer. The protein concentration in the samples was measured by the BCA kit. The DDP content in the samples was determined using HPLC as mentioned above.
Male Balb/c nude mice (∼6 weeks) were obtained from the Institute of Model Animal Wuhan University (Wuhan, China) were raised with standard protocols. All animal‐related experiments were approved by the institutional Ethics Committee of the Changchun National Experimental Animal Center. Each mouse was subcutaneously injected with 6 × 105 A549/DDP cells to establish an A549/DDP tumor‐bearing mouse model.19 Two weeks later, A549/DDP tumor‐bearing mice with a tumor size of ~150 mm3 were taken for in vivo antitumor assays. First mice were randomly divided into four groups (n = 5):
- Saline (as control);
- DDP;
- DDP‐Nsi;
- DDP/OA‐Nsi.
The mice were i.v. administered with the formulations at the dosage of 5 mg/kg DDP every 2 days for seven times. The tumor volumes and mouse weights were recorded each other day.
After the experiment on animal efficacy, the mice were sacrificed, and the tumor tissue of each mouse was excised, fixed in formalin, and cut into 5 μm slices for H&E staining and observed under an optical microscope.
All experiments were performed at least twice and expressed as mean ± SD. Data were analyzed for statistical significance using Student's test. P < 0.05 (*) was considered statistically significant, and P < 0.01 (**) was considered highly significant.20
As given in Table 1, it was observed that the drug‐loading efficiency of DDP in Nsi increased as a function of the charge ratio of DDP, while the particle size of DDP‐Nsi almost remained stable. When the charge ratio increased from 15% to 20%, the drug‐loading efficiency of DDP reached a balance. As a result, we chose the charge ratio of 15% as the optimal ratio for the loading of DDP, and the corresponding drug loading efficiency of DDP was 13.9%. The drug‐loading efficiency of OA also increased as a function of charge ratio without significant changes in particle size. When the charge ratio increased from 8% to 10%, the drug‐loading efficiency of OA reached a balance. As a result, we chose the charge ratio of 8% as the optimal ratio for the loading of OA. Under these specified charge ratios, the drug‐loading efficiency of DDP and OA was 10.8% and 7.6%, respectively, with a final size of 93.5 nm. The weight/weight ratio of DDP and OA in DDP/OA‐Nsi was around 1.4.
TABLECharacterization of various nanocarriers| Number | Charge ratio of DDP (%) | Charge ratio of OA (%) | Drug loading of DDP (%) | Drug loading of OA (%) | Diameter (nm) |
| 1 | 5 | / | 4.8 ± 0.2 | / | / |
| 2 | 10 | / | 9.6 ± 0.3 | / | / |
| 3 | 15 | / | 13.9 ± 0.4 | / | / |
| 4 | 20 | / | 15.1 ± 0.4 | / | / |
| 5 | 15 | 2 | 11.1 ± 0.3 | 1.8 ± 0.1 | 89.3 ± 3.2 |
| 6 | 15 | 5 | 11.1 ± 0.3 | 4.6 ± 0.1 | 92.1 ± 1.6 |
| 7 | 15 | 8 | 10.8 ± 0.3 | 7.6 ± 0.3 | 93.9 ± 2.1 |
| 8 | 15 | 10 | 11.0 ± 0.4 | 7.8 ± 0.3 | 93.5 ± 1.8 |
Abbreviations: DDP, cisplatin; OA, oleanolic acid.
The size distribution of DDP/OA‐Nsi was determined using the dynamic light scattering method. As shown in Figure 1(A), the DDP/OA‐Nsi was narrowly distributed with Gaussian distribution (peak at around 100 nm). It was reported by the previous articles that nanoparticles with a size around 100 nm could take advantage of the EPR effect of the tumor tissue to achieve enhanced accumulation.21,22 Moreover, the stability of nanoparticles was also studied in different conditions (PBS of pH at 7.4 and mouse plasma). As shown in Figure 1(B), DDP/OA‐Nsi remained stable in both PBS and plasma, resulting in only slight fluctuation in size during 6 days of the test, which was beneficial for safe drug delivery as reported by previous articles.23,24
We used the MTT assay to assess the cytotoxicity of different formulations on A549/DDP cells. As shown in Figure 2(A), the IC50 of Nsi was higher than 300 μM in A549/DDP cells, indicating the insignificant cytotoxicity effects of Nsi on A549/DDP cells, which fully proved the biosafety of Nsi. The IC50 of OA‐Nsi was 131 μM, suggesting that OA exerted certain cytotoxicity effects on A549/DDP cells. As for free DDP, DDP‐Nsi, and DDP/OA‐Nsi, as shown in Figure 2(B), free DDP showed the highest IC50 value of 32.1 μM, while DDP/OA‐Nsi showed the lowest IC50 value of 6.1 μM and the DDP‐Nsi exerted a moderate IC50 value of 17.5 μM. It was concluded that DDP/OA‐Nsi showed the best anticancer effects on A549/DDP cells, which might ascribe to the synergistic effect between DDP and OA. As for OA and OA‐Nsi, due to the drug characteristics of OA, the cytotoxicity of both was low, the cytotoxicity of free OA was the lowest.
Next, the flow cytometry was used to detect the apoptosis of A549/DDP cells after treatment of free OA, free DDP, OA‐Nsi, DDP‐Nsi, and DDP/OA‐Nsi or without treatment. The propidium iodide (PI) and Annexin V were selected as fluorescence dyes. As shown in Figure 3, free OA only resulted in 1.8% early apoptosis, the OA‐Nsi resulted in 3.6% apoptosis (including 1.4% early apoptosis and 2.2% late apoptosis), DDP only resulted in 7.6% apoptosis (including 3.0% early apoptosis and 4.6% late apoptosis), respectively. In comparison DDP‐Nsi resulted in 9.9% apoptotic cells (including 2.7% early apoptosis and 7.2% late apoptosis), DDP/OA‐NSI resulted in 15.6% apoptosis (including 6.3% early apoptosis and 9.3% late apoptosis).
These results indicated that the apoptosis‐inducing capacity of OA was weaker than that of DDP. Meanwhile, the cytotoxicity of OA‐Nsi or DDP‐Nsi was stronger than that of the corresponding free drug. This might be due to the superior drug‐loading capacity of Nsi, leading to more intracellular free drug and better apoptotic effect. Moreover, DDP/OA‐Nsi showed better apoptosis‐inducing capacity than DDP‐Nsi or DDP/OA‐Nsi. This might be due to the fact that the co‐delivery of OA and DDP had a synergetic effect on the retention of DDP in cells, which reduced the excretion of DDP and fully realized the apoptosis effect.
The overexpression of MDR‐related proteins, including P‐glycoprotein (P‐gp), is the major mechanism responsible for the MDR of DDP in A549/DDP cells. These proteins can change the uptake and excretion condition of DDP to maintain the intracellular accumulation DDP below the therapeutic concentration. After treating A549/DDP cells with different formulations for different time intervals, the intracellular DDP concentrations were determined. As shown in Figure. 4, after treated with different formulations for 6 h, the intracellular DDP concentrations were not significantly different among the three groups. However, the difference becomes evident as time extended (12 and 24 h). It was observed that the DDP/OA‐Nsi group had the highest intracellular concentration of DDP, followed by DDP‐Nsi and free DDP groups. It was suggested that free DDP was excreted outside A549/DDP cells due to their MDR nature. In contrast, as revealed by many previous studies,25–28 due to the nanoparticle‐mediated endocytosis, which was through specific pathways, the DDP loaded in Nsi can be exempt from the excretion of MDR‐related proteins. However, the different DDP accumulation between DDP‐Nsi and DDP/OA‐Nsi might be due to the fact that OA and DDP were released from the channel structure of Nsi, OA bound to intracellular proteins associated with excretion and occupied the protein binding sites, thus resulting in a decrease in the number of DDP excretion. OA‐mediated synergetic effect on MDR reversion was in line with the previous report29,30 and was beneficial on further increasing the anticancer benefits of the DDS.
Finally, the in vivo anticancer assay was performed using the A549/DDP xenograft tumor model. The tumor volume and body weight of all subjects were recorded during the whole experiment, and the results were shown in Figure 5(A). It was revealed that the tumor volume in the saline group increased more rapidly than other groups. The tumor volume in free DDP grew faster than DDP‐Nsi and DDP/OA‐Nsi groups. In particular, the DDP/OA‐Nsi treatment resulted in the best anticancer outcome, which might be due to the targeting effect of DDP/OA‐Nsi and the synergistic effects between OA and DDP. In addition, the body weight of mice in different groups also showed a significant difference. As shown in Figure 5(B), the body weight of mice in the saline group showed a slight increase, which was similar to that of both DDP‐Nsi and DDP/OA‐Nsi groups. In contrast, the body weight of mice in the free DDP group suffered a significant decline, suggesting that intravenous administration of free DDP exerted strong cytotoxicity on mice, which might be due to the untargeted distribution of drugs. The increased biosafety of DDP‐Nsi and DDP/OA‐Nsi might be due to the nanoparticles‐mediated tumor‐targeting effect, which was in line with the previous report.31
The mice were sacrificed at the end of the experiment, and the tumor tissues were excised. The tumor weights of different groups were shown in Figure 6(A). It was demonstrated that the average tumor weights for different groups were: 3.82 g (saline), 2.11 g (DDP group, achieving a tumor inhibition rate of 44.8%), 1.58 g (DDP‐Nsi group, with a tumor inhibition rate of 58.6%), and 0.98 g (DDP/OA‐Nsi, with the highest tumor inhibition rate of 74.9%). The tumor tissues of mice treated with saline, DDP, DDP‐Nsi, and DDP/OA‐Nsi were exhibited in Figure S1.
The H&E staining of tumor tissues in Figure 6(B) also revealed similar results. It was shown that the saline group exhibited the characteristic tumor cells with an aggressive proliferation manner. In contrast, all DDP‐containing groups showed certain apoptosis effects with cell disruption and debris being observed. In particular, the DDP/OA‐Nsi group showed the most severe cell apoptosis, which was in line with the tumor weight assay in Figure 6(A).
In summary, DDP/OA‐Nsi exhibited much more elevated cytotoxicity than free DDP and DDP‐Nsi, with more apoptosis being observed. In addition, the A549/DDP cells treated with DDP/OA‐Nsi showed more intracellular DDP accumulation than free DDP and DDP‐Nsi, which might be due to the nanoparticle‐assisted endocytosis and OA‐mediated synergistic effects. In the A549/DDP xenograft tumor model, DDP/OA‐Nsi revealed the best anticancer efficacy, which suggested that DDP/OA‐Nsi might be a suitable DDS to solve the MDR in lung cancer therapy.
The authors declare no potential conflict of interest.
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Abstract
Multidrug resistance (MDR) of chemotherapy is one of the significant concerns in cancer therapy. Here in our study, cisplatin (DDP) and oleanolic acid (OA) were co‐loaded in mesoporous silica nanoparticles (Nsi) to construct DDP/OA‐Nsi and solve the DDP‐resistance in lung cancer therapy. The cytotoxicity and apoptosis assays demonstrated that in DDP‐resistant A549/DDP cells, the cytotoxicity of DDP/OA‐Nsi was significantly higher than that of free DDP or DDP single delivery system (DDP‐Nsi). The intracellular drug accumulation study revealed that the intracellular DDP concentration in the DDP/OA‐Nsi group was also higher than that in free DDP and DDP‐Nsi groups. In the A549/DDP xenograft tumor model, DDP/OA‐Nsi showed the best anticancer effect. In summary, DDP/OA‐Nsi was a promising drug delivery system to solve MDR in lung cancer therapy.
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Details
; Li‐Xin Zhang 1 1 Department of Thoracic Oncosurgery‐2, Jilin Province Tumor Hospital, Changchun, China
2 Oral and Maxillofacial Surgery, Jilin Province Tumor Hospital, Changchun, China