Synergistic Combination Chemotherapy of Lung Cancer: Cisplatin and Doxorubicin Conjugated Prodrug Loaded, Glutathione and pH Sensitive Nanocarriers

Introduction

Lung cancer is the leading cause of cancer-related death in the United States and worldwide, and this accounts for about 24% of all cancer deaths in USA.^1,2 Poor diagnosis and limited therapeutic therapies have led to mostly evolved advanced-stage lung cancer patients and lower five-year survival rates (only 19%).³ It is generally known that systemic therapy is the most commonly applied therapeutic regimen for advanced or metastatic lung cancer patients. Whereas, chemotherapy regimens have faced various challenges hindering efficacy, including lack of tumor-targeting, serious toxic effects on healthy tissues, and multidrug resistance.^4,5 Recently, researchers have devoted into new strategies to solve the aforementioned issues containing prodrug technology-based combination therapy, and novel nanoscale targeting approaches (targeting the tumor microenvironment, such as the acidic pH, high concentration of glutathione, etc).^6–9

Prodrug technology-based combination drug therapy has been exploited as a promising treatment strategy to achieve synergistic cancer therapy, minimal drug dose, and decreased side effects.^10,11 Prodrug technology-based combination drug therapy consists of at least three types of systems: two independent anticancer prodrugs; one free anticancer drug and another prodrug; and conjugates composed of two different anticancer drugs.¹¹ In the present study, we synthesized a pH sensitive and glutathione (GSH) sensitive prodrug, cisplatin (CIS) and doxorubicin (DOX) conjugates. CIS is recommended as the first-line therapy for lung cancer by the NCCN guidelines. CIS is a DNA alkylating agent, can bind with genomic DNA and interfere with normal transcription and/or DNA replication, thus triggering toxicity.¹² DOX, a broad-spectrum antitumor chemotherapeutic drug, has been applied in the treatment of a variety of cancers.¹³ CIS has been combined with DOX for lung cancer therapy, showing a significant therapeutic effect.^14–16 As is well known that both CIS and DOX are hydrophobic agents, pH and GSH sensitive CIS-DOX prodrug has the similar properties and could be loaded by nanocarriers and delivered into the tumor site.

For stimuli-responsive nanocarriers, anti-cancer agents can be delivered and release at targeting locations to reach ideal therapeutic anticipation. Among the common stimuli-triggered strategies, pH sensitive is the most frequently researched and used in recent cancer therapy fields.¹⁷ Prodrug-based pH sensitive nanoparticles can be achieved by sensitive chemical linkers such as hydrazone bonds, acetal linkage, etc.^18–20 In our study, DOX and CIS were conjugated by the hydrazone bond and GSH sensitive linker. It is known that the GSH concentration in tumor cells is at least two-folds that for normal cells and intracellular GSH in cisplatin-resistant cells is two-folds as high as that in cisplatin-sensitive cells.^21,22 Therefore, nanocarriers that loaded CIS-DOX pH sensitive and GSH sensitive prodrug (CIS-DOXp) were exploited to realize double efficiencies.

Lipid nanocarriers were designed to encapsulate the previously prepared CIS-DOXp. The drug ratios of CIS to DOX were evaluated by combination index and the physicochemical propriety of CIS-DOXp loaded nanocarriers (CIS-DOXp NC). Release studies were applied in GSH contained medium at different pH values. In vitro cytotoxicity of CIS-DOXp NC was evaluated for lung cancer and drug-resistance lung cancer cells. Tumor-bearing mice were utilized to determine the in vivo anti-cancer efficiency and systemic toxicity drug forms.

Materials and Methods

Materials

CIS, DOX, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), glyceryl monostearate (GMS), and soya lecithin (SL) were obtained from Sigma-Aldrich China (Shanghai, China). Compound 1 (Figure 1) was purchased from TCI China. RPMI Medium 1640, fetal bovine serum (FBS) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Invitrogen Corporation (Carlsbad, CA).

Figure 1 Synthesis of pH and GSH sensitive CIS-DOX prodrug (CIS-DOXp): CIS-DOXp was synthesized by conjugating GSH sensitive CIS prodrug with pH sensitive DOX prodrug.

Synthesis of pH and GSH Sensitive CIS-DOX Prodrug

GSH and pH sensitive CIS-DOX prodrug (CIS-DOXp) was synthesized by conjugating GSH sensitive CIS prodrug with pH sensitive DOX prodrug (Figure 1).

Firstly, GSH sensitive CIS prodrug was synthesized.^23,24 CIS was treated with hydrogen peroxide to obtain Pt(NH₃)₂Cl₂(OH)₂. Pt(NH₃)₂Cl₂(OH)₂ (1 equivalent) was reacted with Compound 1 (2.2 equivalents) overnight to form Pt(NH₃)₂Cl₂(OCOC₆H₁₂COOH)₂.

Secondly, pH sensitive DOX prodrug was synthesized.^20,25 Adipohydrazide (1 equivalent) and EDC (2 equivalents) were reacted in water solution at room temperature for 6 h. DOX (1.2 equivalents) was then added and reacted under acid conditions overnight to form DOX prodrug.

Finally, DOX prodrug (2 equivalents) was activated by EDC (3 equivalents) and Pt(NH₃)₂Cl₂(OCOC₆H₁₂COOH)₂ (1 equivalent) was added and reacted overnight to get CIS-DOXp. The structure of CIS-DOXp was determined by LC-MS and ¹H NMR.

Construction of CIS-DOXp-Loaded Nanocarriers

CIS-DOXp NC was prepared using emulsification and the solvent evaporation method.^26,27 CIS-DOXp (20 mg), GMS (50 mg) and soya lecithin (100 mg) were dissolved in chloroform (10 mL) to produce an oil phase. The oil phase was added to an aqueous phase containing Tween 80 (1.5%) and homogenized (10,000 rpm for 3 min) to get an emulsion. The emulsion was sonicated for 5 min to obtain CIS-DOXp NC.

Non-prodrug contained nanocarriers were prepared using the same method using CIS (5 mg) and DOX (15 mg) instead of CIS-DOXp (20 mg), namely CIS/DOX NC; using CIS (10 mg) instead of CIS-DOXp (20 mg), namely CIS NC; using DOX (30 mg) instead of CIS-DOXp (20 mg), namely DOX NC. Blank nanocarriers (NC) were prepared using the same method without the presence of any drug.

Morphology, Particle Size, and Zeta Potential

The morphology of the CIS-DOXp NC was visualized by transmission electron microscopy (TEM, JEOL 100CX, Tokyo, Japan).²⁸ Particle size, polydispersity index (PDI) and Zeta potential (ζ) of nanocarriers were measured using a Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd, Worcestershire, England) at room temperature.

Colloidal and Serum Stability

The colloidal stability of nanocarriers was determined by measuring the particle size over a 30-day period at two storage conditions: 4°C or 25°C.²⁹ The serum stability of nanocarriers was performed in 50% FBS and incubated at 37°C for various time periods, monitoring the particle size changes.

Drug Loading and Drug Release in vitro

To determine the drug loading, nanocarriers ethanol solution (1 mL) was centrifuged at 15, 000 rpm for 10 min and dissolved in 10 mL KOH (1 M) for 2 h. Then, HCl (10 mL, 1.0 M) was added for neutralizing.³⁰ The suspension was centrifuged at 15, 000 rpm for 10 min, followed by collecting and the upper clear solutions. The drug loading efficiency (LE) and drug loading content (LC) of CIS and DOX were determined by UV-vis spectrometer and ICP-OES. LE and LC were calculated according to the following formulas:

The release of drugs from nanocarriers was evaluated by centrifuging (15, 000 rpm for 10 min) the nanocarriers ethanol solution (0.5 mL).³¹ The resulting solid was dispersed in PBS (15 mL, pH 7.4) with or without GSH (10 mM) at different pH values (pH 7.4 or 5.0). The suspensions were equally distributed to 15 vials (1 mL each) and then incubated at 37°C. One vial of each group was taken out at determined time points and centrifuged (15, 000 rpm for 10 min). The supernatants were taken out and the amount of CIS and DOX was determined by the same method above.

Cells and Animals

Human non-small cell lung cancer cell line: A549 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). CIS resistance lung cancer cell line: A549/CIS cells were provided by Shanghai Ai Research Biological Technology Co., Ltd (Shanghai, China). Cells were cultured in RPMI Medium 1640 supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin at 37°C in an atmosphere of 5% CO₂.

Female BABL/c mice (20 ± 2 g) were provided by Shandong University Laboratory Animal Center (Ji’nan, China) and the mice are maintained and treated in compliance with the policy of the National Institutes of Health guide for the care and use of Laboratory animals and the protocols were approved by the Animal Use and Care Committee of Qilu Hospital of Shandong University.

In vitro Cytotoxicity

The in vitro cytotoxicity of nanocarriers and the corresponding free drugs was examined using the MTT assay.³² A549 cells or A549/CIS cells were seeded in 96-well plates (1000 cells per well) and incubated for 12 h and exposed to CIS-DOXp NC, CIS-DOX NC, CIS NC, DOX NC, and free CIS-DOX. After 48 h, MTT (5 mg/mL, 15 mL) dissolved in phosphate-buffered saline (PBS) was added to each well and incubated for another 3 h, and DMSO (15 mL) was added to each well. The absorbance at 570 nm was measured.

In vitro Synergistic Effect

Synergistic drug combinations are usually evaluated by median-effect analysis.³³ The median-effect method assesses the drug–drug interaction by a term called the “combination index” (CI), which is based on the concentration–response relationship. CI was used to evaluate synergy between CIS and DOX combination against A549/CIS cells, which was performed by CalcuSyn software 1.0. Values of CI < 1, = 1, and > 1 indicate synergy, additivity, and antagonism, respectively.

In vivo Antitumor Effect

A549/CIS cells (100 μL, contained 1 × 10⁶ cells) were subcutaneous injected to the right flank of mice to obtain lung tumor-bearing models.³⁴ When the mean tumor volume reached approximately 100 mm³, tumor-bearing mice were randomly divided into seven treatment groups (eight mice per group), receiving every three days via intravenous tail injection until day 18 with (1) CIS-DOXp NC (CIS 5 mg, DOX 15 mg), (2) CIS-DOX NC (CIS 5 mg, DOX 15 mg), (3) CIS NC (CIS 10 mg), (4) DOX NC (DOX 30 mg), (5) free CIS-DOX (CIS 5 mg, DOX 15 mg), (6) NC, and (7) normal saline (NS). Tumor volume and body weight were measured before each injection. Tumor volume (TV) was calculated by the following formula:

The tumor inhibition rates were calculated according to the tumor volume changes and the tumor images were captured at the end of study.

In vivo Biodistribution

The above-obtained tumor-bearing mice were randomly divided into three treatment groups (eight mice per group), receiving (1) CIS-DOXp NC (CIS 5 mg, DOX 15 mg), (2) CIS-DOX NC (CIS 5 mg, DOX 15 mg), and (3) free CIS-DOX.³⁵ Tumor, heart, liver, spleen, lung, and kidney samples were excised and collected at 2 and 12 h after administration. The tissues were homogenized in lysis buffer, mixed with methanol, and then centrifuged for 30 min. The amount of CIS and DOX was determined by the same method in “Drug loading and drug release in vitro” section.

Statistical Analysis

Data are presented as means ±standard deviation (SD). Statistical analysis was performed by Student’s t-test between two groups, and one-way analysis of variance (ANOVA) among multiple groups. *P < 0.05 was considered statistically significant.

Results

Characterization of CIS-DOXp

CIS-DOXp prodrug was characterized using LC-MS and ¹H NMR. LC-MS (ES+): m/z calculated for C82H108Cl2N12O30Pt: [M + H]+ 2005.6147, found 2005.6325. ¹H NMR (400 MHz, DMSO-d6): δ 1.29, 1. 57, 2.01 and 2.23 belong to CIS prodrug; δ 2.18, 2.82, 3.73, 4.96, 5.01, 7.36 and 8.03 belong to DOX prodrug. The compound yield was 62.3%.

Characterization of CIS-DOXp-Loaded Nanocarriers

The morphology of the CIS-DOXp NC was mostly spherical and uniform (Figure 2A). The size, PDI, zeta potential, CIS LE, and DOX LE were 128.6 ± 3.2 nm, 0.196 ± 0.021, 15.7 ± 1.7 mV, 92.1 ± 2.1%, and 90.4 ± 1.8%, respectively (Table 1). Other nanocarriers showed similar data on these physicochemical properties, indicating the well-designed nature of the systems.

Table 1 Characterization of Nanocarriers (Mean ± SD, n=3)

Figure 2 The morphology of the CIS-DOXp NC (A); the average diameters of nanocarriers monitored at 4°C for 60 days (B); the average diameters of nanocarriers monitored at 25°C for 60 days (Figure 2 (C). The serum stability of nanocarriers performed for 72 h (D). Data presented as mean ±SD, n=8.

Colloidal and Serum Stability

The colloidal stability was determined by monitoring the change in particle size during storage time. The average diameters of nanocarriers were stable when stored at 4°C for 60 days (Figure 2B), in contrast, a remarkably increase of size was exhibited under storage condition of 25°C from day 20 (Figure 2C). The serum stability of nanocarriers was performed for 72 h (Figure 2D). Sizes of nanocarriers remained unchanged over the test period, indicating the good protect ability of the systems from being degraded by the nuclease in the plasma in the administration period.

Drug Release in vitro

CIS-DOXp NC showed different release behaviors in PBS (pH 7.4) with or without the presence of GSH. However, CIS-DOX NC exhibited similar release patterns in both conditions (Figure 3). CIS-DOXp NC and CIS-DOX NC illustrated different release profiles at pH 7.4 or 5.0. More drugs were released form CIS-DOXp NC in pH 5.0 than pH 7.4, but CIS-DOX NC showed no difference between these two pH values (Figure 3).

Figure 3 CIS (A) and DOX (B) release at different pH (7.4 or 5.0) in the medium with or without GSH. Data presented as mean ±SD, n=8, *P < 0.05.

In vitro Cytotoxicity

Cytotoxic effects of CIS-DOXp NC, CIS-DOX NC and free CIS-DOX were different when evaluated on A549 cells or A549/CIS cells. A549 cells were more prominently affected by free CIS-DOX when compared with A549/CIS cells, which attributed to the drug-resistance effect of the latter (Figure 4). CIS-DOXp NC showed slight higher cytotoxicity on A549 cells compared to CIS-DOX NC and free CIS-DOX. However, on A549/CIS cells, significant cell growth inhibition efficacy of CIS-DOXp NC was found than that of CIS-DOX NC (P < 0.05); CIS-DOX NC exhibited remarkable higher cytotoxicity than CIS NC, DOX NC, and free CIS-DOX (P < 0.05).

Figure 4 Cytotoxic effects evaluated on A549 cells (A) or A549/CIS cells (B) by MTT assay. Data presented as mean ±SD, n=8, *P < 0.05.

In vitro Synergistic Effect

In the above section, CIS-DOX NC showed significantly increased cytotoxicity compared with single drug-loaded CIS NC and DOX NC. The A549/CIS cells may have some resistance effect to CIS, so with the increase of DOX amount, the CI values showed obviously reduction (Table 2). The best cell killing ability (the lowest combination index of 0.57) was found at the combination ratio of 1:3 (CIS:DOX, w/w) in the drugs co-loaded formulations, indicating the strongest synergism effect. So this ratio was employed as the amount of drugs in the preparation process.

Table 2 CI Values of Nanocarriers (Mean ± SD, n=3)

In vivo Antitumor Effect

Significant in vivo tumor growth inhibition efficiency was achieved by drugs loaded nanocarriers and free drugs groups in comparison with NS and NC groups (P < 0.05) (Figure 5A). The tumor images also presented similar results (Figure 5B). The tumor volumes of mice treated with CIS-DOXp NC and CIS-DOX NC were more prominently inhibited than free CIS-DOX group, and the best tumor inhibition efficiency was achieved by CIS-DOXp NC group (79.9 ± 3.5%, Table 3). The body weights of nanocarriers treated mice were steady during the study period.

Table 3 Tumor Inhibition Efficiency of Nanocarriers (Mean ± SD, n=3)

Figure 5 Significant In vivo tumor growth inhibition efficiency: tumor volume curves (A), and tumor images (B). Data presented as mean ±SD, n=8, *P < 0.05.

In vivo Biodistribution

Nanocarriers groups distributed relatively higher in tumor, lower in heart and spleen, and relatively high in liver when compared with free CIS-DOX group (Figure 6). At 2 h, the drug accumulation of free CIS-DOX group was higher than 12 h, which may due to the gradual degradation and metabolism in vivo. On the contrary, the biodistribution of nanocarriers increased at 12 h compared with 2 h, suggesting the long-acting ability of the systems.

Figure 6 In vivo CIS (A and B) and DOX (C and D) biodistribution in tumors and other tissues at 2 (A and C) and 12 h (B and D). Data presented as mean ±SD, n=8, *P < 0.05.

Discussion

The tumor microenvironment (TME) is known to encompass unique physicochemical properties that are significantly different from those of the normal tissues. One of the main highlights is generally accepted to be the elevated oxidative stress resulting from the overproduction of reactive oxygen species (ROS) by hypoxic conditions in various TME-associated cells;³⁶ and another is the acidic TME, which is a consequence of accelerated glycolysis and/or oxidative phosphorylation that provide energy and chemical intermediates for cancer cell proliferation.³⁶ The nanocarriers prepared in this study contained GSH and pH sensitive prodrug. The GSH sensitive prodrug-based nanocarriers have been designed by researchers and gained excellent anticancer effect.^37–39 pH sensitive prodrugs have also been utilized to deliver more drugs to the acidic tumor site.^40–42 This research combined the GSH and pH sensitive property to one system. The GSH sensitive property of the nanocarriers was evaluated in PBS with or without the presence of GSH to resemble the difference of cellular environment between tumor cells and normal cells.

Release behaviors of drugs loaded in GSH sensitive nanosystems were developed by researchers.^43–45 Hadipour Moghaddam and colleagues produced glutathione-sensitive nanoparticles for controlled drug delivery:⁴³ The redox responsive particles can release drugs approximately up to 60% in the presence of 10 mM of GSH, while drug release from PBS without GSH was about 15%. Similar results were got by the present study that drug release from CIS-DOXp NC was more sufficient in PBS with than without GSH. pH responsive platforms also showed faster and more release when in acidic pH surroundings.⁴⁶ In the release section, CIS-DOXp NC released faster and more in pH 5.0 than pH 7.4, which is in accordance with the findings of Poudel and Oladipo et al.^47,48

The use of multiple therapeutic agents in combination has become the primary strategy to treat drug-resistant cancers.⁴⁹ There have been several nano-sized vehicles designed for combination drug delivery in cancer chemotherapy.^11,50,51 Wang et al developed a RGD peptide-modified, CIS and paclitaxel co-loaded lipid-polymer nanoparticles for the enhanced lung cancer therapy, which achieved synergistic tumor cells inhibition ability.²⁵ Docetaxel and resveratrol co-encapsulated nanocarriers have been prepared by Song and colleagues and exhibited significant synergistic effects.⁵² In our study, cytotoxic effects of CIS-DOXp NC evaluated on A549/CIS cells illustrated a strong synergism effect according to the term CI introduced by Chou and Talalay in 1984 for quantification of synergism or antagonism for two drugs.⁵³

In tumor tissues, the formation of leaky vessels and pores (100 nm to 2 μm in diameter) and the poor lymphatic system offers great opportunity to treat cancer, which is known as enhanced permeability and retention (EPR) effect.⁵⁴ So the nano-sized carriers could facilitate the delivery of drugs to the tumor site.⁵⁵ Hong et al argued that nanocarriers near 100 nm in diameter tend to represent an optimal range for leveraging the EPR effect and minimizing clearance.⁵⁶ This is in line with our results that CIS-DOXp NC showed a size of 128.6 ± 3.2 nm. The adsorption of proteins on the nanocarriers could cause aggregation, thus leading to an increase in particle size.⁵⁷ Also, the storage stability of nanocarriers was important as disruption of the nanoparticles in the drug delivery system may affect its therapeutic potential.⁵⁸ Sizes of nanocarriers remained unchanged under 4°C and over the test period in serum storage in this research, indicating the high storage stability and good protection ability of the systems from being degraded by the nuclease in the plasma in the administration period. These advantages enable the system to make use of the EPR effect, so as to achieve an enhanced anti-tumor effect and reduce systemic toxicity. In vivo experiments in this paper have shown remarkable antitumor effect and low systemic toxicity. Combined with the system characteristics of combined drug use and double sensitivity, the effectiveness and tolerability of the nano-platform were further illustrated.

Conclusion

In summary, GSH and pH sensitive CIS-DOX prodrug (CIS-DOXp) were synthesized. CIS-DOXp loaded nanocarriers (CIS-DOXp NC) were prepared. CIS-DOXp NC exhibited remarkable cytotoxicity and strong synergism effect. CIS-DOXp NC achieved the best tumor inhibition efficiency in mice with negligible body weight lost, which could be applied as a promising system for the synergistic combination chemotherapy of lung cancer.