Dual-Drug Nanosystem: Etoposide Prodrug and Cisplatin Coloaded Nanostructured Lipid Carriers for Lung Cancer Therapy

Introduction

Lung cancer is the leading cause of cancer death in developed countries, and can be divided into two major classes: non–small cell lung cancer (NSCLC; about 85%) and small-cell lung cancer (about 15%).^1–3 Patients with NSCLC have low overall relative 5-year survival: 25% in the US from 2009 to 2015.⁴ On the basis of clinical studies, the National Comprehensive Cancer Network NSCLC Panel recommends cisplatin (CDDP) combined with docetaxel, etoposide (Etp), gemcitabine, or vinorelbine for preoperative and postoperative chemotherapy.^5–7 Arriagada et al found that CDDP plus Etp in patients with completely resected NSCLC improved patient survival by 56.5%.⁵ Senan et al used Etp 50 mg/m² and CDDP 50 mg/m²intravenously on patients with stage IIIA/B unresectable nonsquamous NSCLC randomly.⁷ Unfortunately, these nonselective combination chemotherapies with multidrug resistance hindered clinical application. Therefore, major efforts have focused on the development of targeted drug-delivery systems based on prodrugs, nanocarriers, and ligand-modified nanoparticles.

CDDP combined with Etp is recommended first-line therapy for NSCLC.⁵ CDDP is a cytotoxic antitumor drug. Its main mechanism involves the binding of genomic DNA in the cell nucleus and interfering with transcription to lead to cell death.⁸ Its main side effects are nephrotoxicity and peripheral neuropathy. Recently, studies reported that targeted nanocarriers could enhance CDDP’s efficacy and reduce its toxicity in healthy cells.^9,10

The topoisomerase II inhibitor Etp inhibits DNA production by affecting the premitotic phase of cell division.¹¹ Poor solubility and chemical instability are major limits to its clinical application.¹² A prodrug is a kind of compound containing a parent drug that carries out biotransformation in vivo through chemical or enzymatic cleavage, and can effectively transfer active molecules.¹³ Prodrug approaches have been developed to modify solubility, increase therapeutic efficacy, and reduce toxicity.¹⁴ Schmidt et al synthesized a glucuronide-based prodrug of Etp that exhibited less cytotoxicity and more water-solubility than Etp itself.¹⁵ In this study, we designed a novel amphiphilic Etp prodrug and tested it on a hydrophobic group (Etp group) and a hydrophilic group (polyethylene glycol, PEG).

Nanoparticles have a length of about 1–100 nm.¹⁶ Nanoparticles are attractive for drug delivery because they have important and unique characteristics, such as much larger surface area:mass ratio than other particles, quantum properties, and the ability to adsorb and carry other compounds.¹⁷ Nanoparticles have been exploited to enhance the pharmacokinetic properties and therapeutic effects of drugs.¹⁸ Lipid nanoparticles are colloidal particles composed of biocompatible and biodegradable lipid matrices, among which nanostructured lipid carriers (NLCs) constituted of blends of lipids in solid and liquid state can be considered the latest generation.¹⁹ NLCs have been engineered to deliver drugs for lung cancer treatment.^20,21 In the present study, NLCs were applied to coload an Etp prodrug (EtpP) and CDDP. EtpP and CDDP coencapsulated in NLCs (EtpP–CDDP NLCs) were prepared and characterized. The in vitro and in vivo antitumor efficiency of EtpP–CDDP NLCs was evaluated and compared with Etp and/or CDDP-loaded NLCs.

Methods

Materials

Etp, CDDP, oleic acid, glyceryl monostearate, coumarin 6 (C₆), and MTT were purchased from Sigma Aldrich (St. Louis, MO). Polyethylene glycol (PEG-NH₂) was purchased from Xi’an Ruixi Biological Technology (Xi’an, China). Other chemicals were of analytical grade or high-performance liquid chromatography grade and used without further purification.

Cells and Animals

Bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B cells), human lung cancer cells (A549 cells), and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, VA). A CDDP-resistant human lung cancer cell line (A549/DDP) was produced by Yiyan Biotechnology (Shanghai, China). Female BALB/c mice derived nu/nu (18–22 g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). A549/DDP cells (10⁶) were injected subcutaneously in the dorsal skin of BALB/c mice. The tumors were grown to around 100 mm³ to obtain mice bearing A549/DDP cell xenografts. All animal experiments were approved by the Animal Ethics Committee of Wuxi Dashan Medical Beauty Clinic and followed the policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Synthesis of Etp Prodrug

EtpP was synthesized by linking the phenolic hydroxyl group of Etp with PEG (Figure 1).²² In brief, PEG-NH₂ (1 equivalent) and phosgene (1.5 equivalents) were dissolved in CH₂Cl₂ (10 mL) at 0°C, then triethylamine (TEA, 3 equivalents) was added dropwise to get PEG-NH-COCl. DMAP (1.2 equivalents) was added to PEG-NH-COCl, followed by adding Etp (1.2 equivalents) in CH₂Cl₂ (10 mL). The mixture was stirred (60 min) and the solvent removed under reduced pressure. The product was purified by column chromatography on silica gel (CH₂Cl₂/CH₃CN: 8/2). A white solid (77%) was isolated to achieve EtpPEG (EtpP). ¹H NMR of EtpPEG in dimethyl sulfoxide-d₆ at 300 MHz: δ 8.21 and 7.38 (–NH–C(=O)–), 6.72–6.01 (belonging to Etp), 4.12 (–O–CH₂–C(=O)–N–), 3.59–3.81 (belonging to PEG), 1.95 (3, –CH₂–C(=O)–N–), 1.26 (–CH₂–), 0.96 (–CH₃).

Figure 1 The Etp prodrug was synthesized by linking the phenolic hydroxyl group of Etp with PEG. Note: ETP prodrug was synthesized by linking the phenolic hydroxyl group of ETP with PEG.

Preparation of EtpP–CDDP NLCs

EtpP–CDDP NLCs were prepared using film ultrasound.²³ Briefly, an oil phase was formed by dissolving EtpP (20 mg), CDDP (10 mg), oleic acid (90 mg), and glyceryl monostearate (180 mg) in CH₂Cl₂ (20 mL) under a constant stirring at 400 rpm. The aqueous phase was created by dissolving poloxamer 188 in pure water at 50°C. The oil phase was added dropwise to the aqueous phase and the organic solvent removed at 50°C under vacuum using a rotary evaporator. Then, the mixture was ultrasonically dispersed at 50°C and cooled to room temperature to obtain EtpP–CDDP NLCs (Figure 2). Etp (not prodrug) and CDDP coloaded NLCs (Etp–CDDP NLCs) were prepared by the same method using Etp and PEG (20 mg) instead of EtpP (20 mg). EtpP- or CDDP-loaded NLCs (EtpP NLCs or CDDP NLCs) were prepared by the same method using Etp (20 mg) or CDDP (10 mg), without adding another drug. Blank NLCs were prepared by the same method using no drug.

Figure 2 EtpP–CDDP NLCs and TEM. EtpP–CDDP NLCs were prepared using film ultrasound. The morphology of EtpP–CDDP NLCs was examined using transmission electron microscopy (TEM). Note: ETP-P/CDDP NLCs were prepared using film-ultrasonic method.

Particle Size, -Potential, and Stability

Particle sizes, polydispersity index (PDI) values, and -potential of NLCs were determined using a laser light scattering.²⁴ Morphology of EtpP–CDDP NLCs was examined using transmission electron microscopy. A drop of EtpP–CDDP NLC suspension was placed on a copper grid and air-dried, followed by negative staining with one drop of 3% aqueous solution of sodium phosphotungstate. Photos were taken after air-drying. The stability of NLCs was checked in PBS at 2°C–8°C and cell-culture medium (DMEM + 10% FBS) at 37°C. Changes in particle size and appearance were recorded over time.

Drug Loading and In Vitro Drug Release

Drug-encapsulation efficiency (EE) and -loading capacity (LC) of NLCs were detected by separating the drugs from NLCs under centrifugation (10,000 rpm for 10 min). The amount of loaded drugs in NLCs was determined by UV–visible spectroscopy using a UV spectrophotometer.²⁵ The selected wavelengths used for the measurement of Etp and CDDP were 254 nm and 210 nm, respectively.^9,25 EE and LC were calculated by:

EE (%) = amount of entrapped drugs/total weight of drugs × 100.

LC (%) = amount of drugs in nanoparticles/mass of nanoparticles × 100.

In vitro release of drugs from NLCs was analysed using dialysis at 37°C.^26,27 In brief, NLCs (2 mL) were put into a dialysis bag and immersed in 100 mL PBS in the presence of 10% FBS (pH 7.4) and placed on a shaking bed at 37°C with a rotation speed of 100 rpm. Samples (2 mL) were withdrawn at predetermined time intervals and replaced by 2 mL fresh release medium. The released drugs were analyzed by the same method as previously mentioned.

Cellular Uptake

Cellular uptake of NLCs was visualized using an inversion fluorescence microscope (CKX53; Olympus, Tokyo, Japan) and quantified by fluorescence-activated cell sorting.²⁸ C₆ was used as the fluorescent probe and C₆-containing NLCs were prepared by the same method, adding C₆ (5 mg) along with doxorubicin in the oil phase. A549/DDP cells were seeded in 24-well culture plates (5×10⁴ cells per well) and incubated with various formulations (200 μg/mL) for 2 h. After incubation, cells were washed three times with D-Hank’s solution, photographed with the inversion fluorescence microscope, and quantified using a fluorescence-activated cell sorter (FACS, Becton, Dickinson, Franklin Lakes, NJ, λ_excitation 430 nm, λ_emission 485 nm).

Cytotoxicity and Synergistic Effect

Cytotoxicity of drugs and drug-containing NLCs was assessed by evaluating cell viability using MTT assays.²⁹ A549/DDP cells, BEAS-2B, cells or HUVECs (2×10⁴ cells/well) were seeded in 96-well plates and allowed to grow for 24 h, then were treated with EtpP–CDDP NLCs, Etp–CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP–CDDP, free EtpP, and free CDDP at various drug concentrations. After 48 h of incubation, the medium in each well was replaced with fresh medium (100 µL), 10 µL MTT solution (5 mg/mL) added to each well (10% v/v), and cells further incubated for 4 hours at 37°C. DMSO (150 µL) was added to each well after the removal of medium and shaken for 10 minutes. A microplate reader was utilized to record absorbance at a wavelength of 570 nm. Mean drug concentration required for 50% growth inhibition (IC₅₀) was calculated.

An effective method to evaluate synergistic drug combinations in vitro is median-effect analysis, introduced by Chou and Talalay.³⁰ The median-effect method assesses drug–drug interaction using a “combination index” (CI), which is based on the concentration–response relationship.³¹ CI < 1 represents synergism and CI > 1 represents antagonism. In this study, values were calculated by CI₅₀ = (concentration of Etp in combination system)/(IC₅₀ of Etp) + (concentration of CDDP in combination system)/(IC₅₀ of CDDP). Caspase 3 activity assays were carried out according to the instructions of the manufacturer with a caspase 3 activity kit (Beyotime, Shanghai, China).^32,33 After treatment with EtpP–CDDP NLCs, Etp–CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP–CDDP, free EtpP, and free CDDP (100 µg/mL for 24 h), A549/DDP cells were washed, collected, lysed, centrifuged, and analyzed for total protein by a SpectraMax M2 microplate reader (Molecular Devices, USA) at 405 nm.

In Vivo Pharmacokinetics and Tissue Distribution

BALB/c nude mice were injected with A549/DDP cells in the right flank to produce lung cancer–bearing xenografts. When tumors had grown to a volume of about 100 mm³, the mice were randomly divided into three groups (six in each group). EtpP–CDDP NLCs, Etp–CDDP NLCs, and free EtpP–CDDP were intravenously injected in mice at a drug dose of 5 mg/kg body weight.^34,35 At determined time points, blood (500 μL) was collected by cardiac puncture after euthanizing the mice. Plasma was separated by centrifuging samples (2500 rpm, 15 min). At 1 h and 48 h, the tumor tissue and other main tissue types (heart, liver, spleen, lung, and kidney) were removed, washed, and homogenized. The mixture was vortexed and centrifuged (15,000 rpm, 10 min), and the supernatants along with the plasma concentration of drugs were determined by the same method as in the Drug Loading and In Vitro Drug Release section.

In Vivo Toxicity and Antitumor Ability

Lung cancer–bearing xenografts were randomly divided into nine groups (six in each group). EtpP–CDDP NLCs, Etp–CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP–CDDP, free EtpP, free CDDP, and 0.9% saline were intravenously injected in the tail vein every 3 days.³⁶ The sizes of the tumors were measured using calipers before every injection, and tumor volume was calculated by long axis × (short axis)²/2. In vivo toxicity was observed by monitoring changes in the body weights of mice every 3 days and white blood cells (WBCs), alanine aminotransferase (Alt; liver function), and creatinine (Cre; kidney function).

Statistical Analysis

The sample size in all experiments was at least six (n≥6). Data distribution was assessed with Shapiro–Wilk tests and QQ plots. Statistical analysis was performed using unpaired t-tests (between two groups) and one-way ANOVA (among three or more groups), followed by Tukey’s post hoc test using SPSS 20.0. Statistical significance was taken as P<0.05. Data are presented as means ± SD.

Results

Characterization of EtpP–CDDP NLCs

EtpP–CDDP NLCs showed a uniformly spherical morphology (Figure 2) with a size of 176.8±4.9 nm and -potential of –31.9±3.2 mV (Table 1). The EE of NLCs was around 90% and that of LCs 3.3%–5.4%. The stability of NLCs was evaluated by changes in size, presented in Figure 3. NLC formulations showed good stability during 90 days of storage in PBS at 2°C–8°C, while NLCs remained stable in the first 6 days (Figure 4). In vitro drug release of both EtpP and CDDP from LPNs was sustained. The drugs had completed their release from NLCs by 48 h, which could be a reference for in vitro and in vivo antitumor studies.

Table 1 Characterization of NLCs (means ± SD, n=8)

Figure 3 The stability of NLCs evaluated in PBS at 2°C–8°C (A) and cell-culture medium (DMEM + 10% FBS) at 37°C (B) by changes in size with time. Note: Data presented as means ± SD, n=6.

Figure 4 In vitro release of Etp (A) and CDDP (B) from NLCs was analyzed using dialysis. NLCs (2 mL) were put in a dialysis bag, immersed in 100 mL PBS in the presence of 10% FBS (pH 7.4), and placed on a shaking bed at 37°C with a rotation speed of 100 rpm. Note: Data presented as means ± SD, n=6.

Cellular Uptake

Figure 5 illustrate the cellular uptake efficiency of EtpP–CDDP NLCs, Etp–CDDP NLCs and blank NLCs: 57.4%±3.9%, 55.1%±4.1% and 59.2%±3.5% into A549/DDP cells, respectively. This could be recognized as high cellular uptake of the carriers, as illustrated by Hong et al and Pang et al^28,37

Figure 5 Cellular uptake efficiency of EtpP–CDDP NLCs, Etpp–CDDP NLCs, and blank NLCs. Uptake of NLCs was visualized using inversion fluorescence microscopy and quantified by fluorescence-activated cell sorting. Note: Data presented as means ± SD, n=6.

Cytotoxicity and Synergistic Effect

Blank NLCs did not change cell viability, while free drug and drug-loaded NLCs showed obvious cell-inhibition efficiency compared with the control (P<0.05) EtpP–CDDP NLCs exhibited higher cytotoxicity than Etp–CDDP NLCs, EtpP NLCs, and free EtpP–CDDP on A549/DDP cells (Figure 6A and Supplementary Figure 1, P<0.05). On the contrary, drug-loaded NLCs did not exhibit enhanced cell inhibition on human normal lung epithelial cells (BEAS-2B cells) or HUVECs compared with free drugs (Figure 6B and C). The CI₅₀ values that illustrated the synergistic effect of the dual drug–containing systems are presented in Figure 6D, which shows that CI values were <1 with fraction of affected cells (Fa) of 20%–80% (0.2–0.8). Caspase 3 activity assays showed that caspase activity in A549/DDP cells treated with EtpP–CDDP NLCs increased significantly in comparison to Etp–CDDP NLCs, EtpP NLCs, and free EtpP–CDDP (P<0.05). This indicated that EtpP–CDDP NLCs promoted caspase 3 activation in lung cancer cells (Figure 6E).

Figure 6 Cytotoxicity of drugs and drug-containing NLCs assessed by evaluating A549/DDP (A), BEAS-2B (B), and HUVECs (C) viability using MTT assays. A549/DDP and BEAS-2B cells (2×104 cells/well) were seeded in 96-well plates and allowed to grow for 24 h, then were treated with EtpP–CDDP NLCs, Etpp–CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP–CDDP, free EtpP, and free CDDP at various drug concentrations. CI50 values illustrated the synergistic effect of the dual drug–containing systems (D). Caspase 3 activity assays were carried out according to the instructions of the manufacturer with a caspase 3 activity kit (E). *P<0.05. Note: Data presented as means ± SD, n=6.

In Vivo Pharmacokinetics and Tissue Distribution

The blood concentration–time profiles of EtpP–CDDP NLCs, Etp–CDDP NLCs, and free EtpP–CDDP are presented in Figure 7. EtpP–CDDP NLCs and Etp–CDDP NLCs exhibited more sustained plasma retention effects than the rapidly decreasing concentration of free EtpP–CDDP. Tissue biodistribution of drugs is summarized in Figure 8. At both 1 and 48 h after intravenous injection, EtpP–CDDP NLCs showed higher drug distribution in tumors than Etp–CDDP NLCs and free EtpP–CDDP (Supplementary Figures 2 and 3, P<0.05). Etp–CDDP NLCs exhibited higher tumor drug accumulation than free EtpP–CDDP (P<0.05). Tumor accumulation of free EtpP–CDDP decreased rapidly after 48 h, while NLC formulations remained at high levels at 48 h.

Figure 7 Etp (A) and CDDP (B) blood concentration–time profiles of EtpP–CDDP NLCs, Etpp–CDDP NLCs, and free EtpP–CDDP. Mixtures were vortexed and centrifuged (15,000 rpm, 10 min), and supernatants and plasma concentration of drugs were determined. Note: Data presented as means ± SD, n=6.

Figure 8 Tissue Etp (A) and CDDP (B) biodistribution of EtpP–CDDP NLCs, Etpp–CDDP NLCs, and free EtpP–CDDP. At 1 and 48 h, the tumor tissue and other main tissue types (heart, liver, spleen, lung, and kidney) were removed, washed, homogenized, and analyzed. *P<0.05. Note: Data presented as means ± SD, n=6.

In Vivo Antitumor Ability

In vivo antitumor ability of drug-loaded NLCs and free drugs were investigated on lung cancer–bearing mice (Figure 9). Drug-loaded NLC groups showed remarkably higher tumor inhibition compared to free drugs (Figure 9A and Supplementary Figure 4, P<0.05). Dual drug–loaded EtpP–CDDP NLCs exhibited higher inhibit rates than single drug–loaded NLCs (P<0.05). Most importantly, EtpP–CDDP NLCs illustrated enhanced antitumor efficacy than non-prodrug–containing Etp–CDDP NLCs (P<0.05). Figure 9B shows that drug-containing NLCs did not cause changes in body weight, while free drugs caused a decrease in weight (Supplementary Figure 5, P<0.05). An increase in Cre was observed in the free-drug groups (Figure 9C), while the Alt and WBC values of NLCs and free drugs were not obviously changed (Figure 9A and B).

Figure 9 In vivo antitumor activity (A), body-weight changes (B), WBC (C), Alt (D), and Cre (E) in lung cancer–bearing mice. Tumor sizes were measured using calipers before every injection and tumor volume calculated by long axis × (short axis)2/2. Body-weight changes were monitored every 3 days to evaluate systemic toxicity. *P<0.05. Note: Data presented as means ± SD, n=6.

Discussion

At the beginning of this study, an Etp prodrug was synthesized. Under controlled conditions (DMAP and CH₂Cl₂), the alcohol groups of the Etp glucose moiety did not react, and only the –OH phenol group was coupled with Etp.²⁶ After that, dual drug–loaded NLCs were prepared. NLCs have been reported to be nanosized with narrow size distribution and high drug EE.³⁸ The size of the prepared NLCs was around 170 nm, which proved to be efficiently internalized into cells.³⁹ The EE of NLCs was about 90%, which promised good loading ability of the systems.⁴⁰

The release profiles of the NLCs revealed a two-stage process, with relatively fast drug release in the initial stage and slow release subsequently, which was also described by Luan et al.⁴¹ The biphasic drug-release patterns in that study were described as fast release in the initial 12 h, about 50%, followed by sustained release 30% of the remaining by 48 h. This phenomenon was caused by the drug concentration gradient between the nanoparticle and the medium: a burst release of the drug dispersed on the surface of NLCs in the initial stage, then gradual release due to the amount of drug in the nanoparticles depleting with the release process.⁴¹ Cellular uptake of nanoparticles can be quantitatively detected by the fluorescence method.³⁹ Cellular uptake efficiency of NLCs was >50%, which may be recognized as relatively high.^33,37

Nanoparticulate drug-delivery systems may induce significant cytotoxic effects in systems, because free drugs diffuse through cell membranes, but nanoparticles are internalized through the endocytic pathway, resulting in greater uptake and higher cytotoxicity than free drugs.⁴² EtpP–CDDP NLCs exhibited higher cytotoxicity than Etp–CDDP NLCs, EtpP NLCs and free EtpP–CDDP on A549/DDP cells. This may prove the enhanced ability of the NLCs, which were able to adhere to the cell membrane due to the similar nature of the lipids and the cell membrane.⁴³ This characteristic may enhance intracellular drug accumulation and perform better in cancer therapy. Combination therapy using nanoparticles containing CDDP has received much interest in cancer therapy to overcome CDDP-resistan, eg, Liang et al developed a CDDP and vinorelbine coencapsulated nano-platform for lung cancer treatment.⁴⁴ When combination therapy is used in the system, evaluation of the synergistic effect is important, and CI analyses are one of the most reliable methods.⁴⁵ The CI₅₀ values of the systems tested in this research were <1, which illustrated the synergistic effect of the dual drug–containing systems.

In vivo pharmacokinetic and tissue-distribution studies were carried out on lung cancer–bearing mice. Higher accumulation in tumor tissue may contribute to the passive targeting ability of nanoparticles through the enhanced permeability-and-retention (EPR) effect.⁴⁶ In vivo tumor-inhibition results showed that blank NLCs had similar tumor-growth curves as controls, suggesting that nanomaterials without drug could not inhibit tumor growth.¹⁹ EtpP–CDDP NLCs showed the most significant antitumor efficiency (P=0.0186), better than non-prodrug–containing Etp–CDDP NLCs. This result is in accordance with the findings of Wang et al that prodrug-based systems can improve lung cancer chemotherapy.⁴⁷ Mice treated with nanoparticles showed negligible changes in Cre, Alt, and WBCs over the control group, while free drugs affected some parameters. No obvious weight loss was observed in any of the test NLC groups, indicating good tolerance of the systems, which was also reported by Zhang et al, ie, the lipid structure of NLCs has high affinity with the lipid cell surface, promotes the fusion of carriers to cells, and thus delivers the drug without high systemic toxicity.⁴⁸

Conclusion

An Etp prodrug was synthesized and EtpP–CDDP NLCs prepared. EtpP–CDDP NLCs exhibited high tumor-cell uptake, high cytotoxicity, sustained plasma retention effect, increased accumulation in tumor tissue, and improved tumor-inhibition efficiency. These characteristics could make this a promising drug-delivery system for lung cancer combination therapy.