{"id":28231,"date":"2022-12-05T08:10:00","date_gmt":"2022-12-05T09:10:00","guid":{"rendered":"https:\/\/peymantaeidi.net\/stem-cell\/?p=28231"},"modified":"2022-12-05T09:44:36","modified_gmt":"2022-12-05T09:44:36","slug":"dual-drug-nanosystem-etoposide-prodrug-and-cisplatin-coloaded-nanostructured-lipid-carriers-for-lung-cancer-therapy","status":"publish","type":"post","link":"https:\/\/peymantaeidi.net\/stem-cell\/2022\/12\/05\/dual-drug-nanosystem-etoposide-prodrug-and-cisplatin-coloaded-nanostructured-lipid-carriers-for-lung-cancer-therapy\/","title":{"rendered":"Dual-Drug Nanosystem: Etoposide Prodrug and Cisplatin Coloaded Nanostructured Lipid Carriers for Lung Cancer Therapy"},"content":{"rendered":"

1<\/sup>Department of Pharmacy, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu Province, 214000, People\u2019s Republic of China; 2<\/sup>Department of Pharmacy, Wuxi Dashan Medical Beauty Clinic, Wuxi, Jiangsu Province, 214001, People\u2019s Republic of China<\/p>\n

Purpose:<\/strong> Cisplatin (CDDP) and etoposide (Etp) are recommended first-line therapy for lung cancer. Nanostructured lipid carriers (NLCs) are engineered to deliver drugs for lung cancer treatment. In the present study, NLCs were applied to coload an Etp prodrug (EtpP) and CDDP.
Methods:<\/strong> The Etp prodrug was synthesized by linking the phenolic hydroxyl group of Etp with polyethylene glycol (PEG). EtpP and CDDP coencapsulated NLCs (EtpP\u2013CDDP NLCs) were prepared using film ultrasound. Cytotoxicity of drugs and drug-containing NLCs was assessed by evaluating cell viability using MTT assays. In vivo antitumor efficiency of EtpP\u2013CDDP NLCs was evaluated on lung cancer\u2013bearing xenografts.
Results:<\/strong> EtpP\u2013CDDP NLCs showed a uniformly spherical morphology with a size of 176.8\u00b1 4.9 nm and -potential of \u2013 31.9\u00b1 3.2 mV. Cellular uptake efficiency of EtpP\u2013CDDP NLCs was 57.4%\u00b1 3.9% on A549\/DDP cells. EtpP\u2013CDDP NLCs exhibited more sustained plasma retention, the highest drug distribution in tumors, and the highest tumor-inhibition rates in lung tumor\u2013bearing mice.
Conclusion:<\/strong> EtpP\u2013CDDP NLCs improved tumor-cell uptake, cytotoxicity, and tumor-inhibition efficiency, and could be used as a promising drug-delivery system for lung cancer combination therapy.<\/p>\n

Keywords:<\/strong> lung cancer, prodrug, etoposide, cisplatin, nanostructured lipid carriers<\/p>\n

\n
\n

Introduction<\/h2>\n

Lung cancer is the leading cause of cancer death in developed countries, and can be divided into two major classes: non\u2013small cell lung cancer (NSCLC; about 85%) and small-cell lung cancer (about 15%).1\u20133<\/a><\/sup> Patients with NSCLC have low overall relative 5-year survival: 25% in the US from 2009 to 2015.4<\/a><\/sup> 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\u20137<\/a><\/sup> Arriagada et al found that CDDP plus Etp in patients with completely resected NSCLC improved patient survival by 56.5%.5<\/a><\/sup> Senan et al used Etp 50 mg\/m2<\/sup> and CDDP 50 mg\/m2<\/sup>intravenously on patients with stage IIIA\/B unresectable nonsquamous NSCLC randomly.7<\/a><\/sup> 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.<\/p>\n

CDDP combined with Etp is recommended first-line therapy for NSCLC.5<\/a><\/sup> 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<\/a><\/sup> Its main side effects are nephrotoxicity and peripheral neuropathy. Recently, studies reported that targeted nanocarriers could enhance CDDP\u2019s efficacy and reduce its toxicity in healthy cells.9<\/a>,10<\/a><\/sup><\/p>\n

The topoisomerase II inhibitor Etp inhibits DNA production by affecting the premitotic phase of cell division.11<\/a><\/sup> Poor solubility and chemical instability are major limits to its clinical application.12<\/a><\/sup> 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<\/a><\/sup> Prodrug approaches have been developed to modify solubility, increase therapeutic efficacy, and reduce toxicity.14<\/a><\/sup> Schmidt et al synthesized a glucuronide-based prodrug of Etp that exhibited less cytotoxicity and more water-solubility than Etp itself.15<\/a><\/sup> 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).<\/p>\n

Nanoparticles have a length of about 1\u2013100 nm.16<\/a><\/sup> 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<\/a><\/sup> Nanoparticles have been exploited to enhance the pharmacokinetic properties and therapeutic effects of drugs.18<\/a><\/sup> 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<\/a><\/sup> NLCs have been engineered to deliver drugs for lung cancer treatment.20<\/a>,21<\/a><\/sup> In the present study, NLCs were applied to coload an Etp prodrug (EtpP) and CDDP. EtpP and CDDP coencapsulated in NLCs (EtpP\u2013CDDP NLCs) were prepared and characterized. The in vitro and in vivo antitumor efficiency of EtpP\u2013CDDP NLCs was evaluated and compared with Etp and\/or CDDP-loaded NLCs.<\/p>\n

Methods<\/h2>\n

Materials<\/h3>\n

Etp, CDDP, oleic acid, glyceryl monostearate, coumarin 6 (C6<\/sub>), and MTT were purchased from Sigma Aldrich (St. Louis, MO). Polyethylene glycol (PEG-NH2<\/sub>) was purchased from Xi\u2019an Ruixi Biological Technology (Xi\u2019an, China). Other chemicals were of analytical grade or high-performance liquid chromatography grade and used without further purification.<\/p>\n

Cells and Animals<\/h3>\n

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\u201322 g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). A549\/DDP cells (106<\/sup>) were injected subcutaneously in the dorsal skin of BALB\/c mice. The tumors were grown to around 100 mm3<\/sup> 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<\/em>.<\/p>\n

Synthesis of Etp Prodrug<\/h3>\n

EtpP was synthesized by linking the phenolic hydroxyl group of Etp with PEG (Figure 1<\/a>).22<\/a><\/sup> In brief, PEG-NH2<\/sub> (1 equivalent) and phosgene (1.5 equivalents) were dissolved in CH2<\/sub>Cl2<\/sub> (10 mL) at 0\u00b0C, 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 CH2<\/sub>Cl2<\/sub> (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 (CH2<\/sub>Cl2<\/sub>\/CH3<\/sub>CN: 8\/2). A white solid (77%) was isolated to achieve EtpPEG (EtpP). 1<\/sup>H NMR of EtpPEG in dimethyl sulfoxide-d<\/em>6<\/sub> at 300 MHz: \u03b4 8.21 and 7.38 (\u2013NH\u2013C(=O)\u2013), 6.72\u20136.01 (belonging to Etp), 4.12 (\u2013O\u2013CH2<\/sub>\u2013C(=O)\u2013N\u2013), 3.59\u20133.81 (belonging to PEG), 1.95 (3, \u2013CH2<\/sub>\u2013C(=O)\u2013N\u2013), 1.26 (\u2013CH2<\/sub>\u2013), 0.96 (\u2013CH3<\/sub>).\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 1<\/strong> The Etp prodrug was synthesized by linking the phenolic hydroxyl group of Etp with PEG.<\/p>\n

Note<\/strong>: ETP prodrug was synthesized by linking the phenolic hydroxyl group of ETP with PEG.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Preparation of EtpP\u2013CDDP NLCs<\/h3>\n

EtpP\u2013CDDP NLCs were prepared using film ultrasound.23<\/a><\/sup> Briefly, an oil phase was formed by dissolving EtpP (20 mg), CDDP (10 mg), oleic acid (90 mg), and glyceryl monostearate (180 mg) in CH2<\/sub>Cl2<\/sub> (20 mL) under a constant stirring at 400 rpm. The aqueous phase was created by dissolving poloxamer 188 in pure water at 50\u00b0C. The oil phase was added dropwise to the aqueous phase and the organic solvent removed at 50\u00b0C under vacuum using a rotary evaporator. Then, the mixture was ultrasonically dispersed at 50\u00b0C and cooled to room temperature to obtain EtpP\u2013CDDP NLCs (Figure 2<\/a>). Etp (not prodrug) and CDDP coloaded NLCs (Etp\u2013CDDP 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.\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 2<\/strong> EtpP\u2013CDDP NLCs and TEM. EtpP\u2013CDDP NLCs were prepared using film ultrasound. The morphology of EtpP\u2013CDDP NLCs was examined using transmission electron microscopy (TEM).<\/p>\n

Note<\/strong>: ETP-P\/CDDP NLCs were prepared using film-ultrasonic method.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Particle Size, \"image\"<\/span>-Potential, and Stability<\/h3>\n

Particle sizes, polydispersity index (PDI) values, and \"image\"<\/span>-potential of NLCs were determined using a laser light scattering.24<\/a><\/sup> Morphology of EtpP\u2013CDDP NLCs was examined using transmission electron microscopy. A drop of EtpP\u2013CDDP 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\u00b0C\u20138\u00b0C and cell-culture medium (DMEM + 10% FBS) at 37\u00b0C. Changes in particle size and appearance were recorded over time.<\/p>\n

Drug Loading and In Vitro Drug Release<\/h3>\n

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\u2013visible spectroscopy using a UV spectrophotometer.25<\/a><\/sup> The selected wavelengths used for the measurement of Etp and CDDP were 254 nm and 210 nm, respectively.9<\/a>,25<\/a><\/sup> EE and LC were calculated by:<\/p>\n

EE (%) = amount of entrapped drugs\/total weight of drugs \u00d7 100.<\/p>\n

LC (%) = amount of drugs in nanoparticles\/mass of nanoparticles \u00d7 100.<\/p>\n

In vitro release of drugs from NLCs was analysed using dialysis at 37\u00b0C.26<\/a>,27<\/a><\/sup> 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\u00b0C 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.<\/p>\n

Cellular Uptake<\/h3>\n

Cellular uptake of NLCs was visualized using an inversion fluorescence microscope (CKX53; Olympus, Tokyo, Japan) and quantified by fluorescence-activated cell sorting.28<\/a><\/sup> C6<\/sub> was used as the fluorescent probe and C6<\/sub>-containing NLCs were prepared by the same method, adding C6<\/sub> (5 mg) along with doxorubicin in the oil phase. A549\/DDP cells were seeded in 24-well culture plates (5\u00d7104<\/sup> cells per well) and incubated with various formulations (200 \u03bcg\/mL) for 2 h. After incubation, cells were washed three times with D-Hank\u2019s solution, photographed with the inversion fluorescence microscope, and quantified using a fluorescence-activated cell sorter (FACS, Becton, Dickinson, Franklin Lakes, NJ, \u03bbexcitation<\/sub> 430 nm, \u03bbemission<\/sub> 485 nm).<\/p>\n

Cytotoxicity and Synergistic Effect<\/h3>\n

Cytotoxicity of drugs and drug-containing NLCs was assessed by evaluating cell viability using MTT assays.29<\/a><\/sup> A549\/DDP cells, BEAS-2B, cells or HUVECs (2\u00d7104<\/sup> cells\/well) were seeded in 96-well plates and allowed to grow for 24 h, then were treated with EtpP\u2013CDDP NLCs, Etp\u2013CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP\u2013CDDP, 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 \u00b5L), 10 \u00b5L MTT solution (5 mg\/mL) added to each well (10% v\/v), and cells further incubated for 4 hours at 37\u00b0C. DMSO (150 \u00b5L) 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<\/sub>) was calculated.<\/p>\n

An effective method to evaluate synergistic drug combinations in vitro is median-effect analysis, introduced by Chou and Talalay.30<\/a><\/sup> The median-effect method assesses drug\u2013drug interaction using a \u201ccombination index\u201d (CI), which is based on the concentration\u2013response relationship.31<\/a><\/sup> CI < 1 represents synergism and CI > 1 represents antagonism. In this study, values were calculated by CI50<\/sub> = (concentration of Etp in combination system)\/(IC50<\/sub> of Etp) + (concentration of CDDP in combination system)\/(IC50<\/sub> 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<\/a>,33<\/a><\/sup> After treatment with EtpP\u2013CDDP NLCs, Etp\u2013CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP\u2013CDDP, free EtpP, and free CDDP (100 \u00b5g\/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.<\/p>\n

In Vivo Pharmacokinetics and Tissue Distribution<\/h3>\n

BALB\/c nude mice were injected with A549\/DDP cells in the right flank to produce lung cancer\u2013bearing xenografts. When tumors had grown to a volume of about 100 mm3<\/sup>, the mice were randomly divided into three groups (six in each group). EtpP\u2013CDDP NLCs, Etp\u2013CDDP NLCs, and free EtpP\u2013CDDP were intravenously injected in mice at a drug dose of 5 mg\/kg body weight.34<\/a>,35<\/a><\/sup> At determined time points, blood (500 \u03bcL) 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.<\/p>\n

In Vivo Toxicity and Antitumor Ability<\/h3>\n

Lung cancer\u2013bearing xenografts were randomly divided into nine groups (six in each group). EtpP\u2013CDDP NLCs, Etp\u2013CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP\u2013CDDP, free EtpP, free CDDP, and 0.9% saline were intravenously injected in the tail vein every 3 days.36<\/a><\/sup> The sizes of the tumors were measured using calipers before every injection, and tumor volume was calculated by long axis \u00d7 (short axis)2<\/sup>\/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).<\/p>\n

Statistical Analysis<\/h3>\n

The sample size in all experiments was at least six (n\u22656). Data distribution was assessed with Shapiro\u2013Wilk tests and QQ plots. Statistical analysis was performed using unpaired t<\/em>-tests (between two groups) and one-way ANOVA (among three or more groups), followed by Tukey\u2019s post hoc test using SPSS 20.0. Statistical significance was taken as P<\/em><0.05. Data are presented as means \u00b1 SD.<\/p>\n

Results<\/h2>\n

Characterization of EtpP\u2013CDDP NLCs<\/h3>\n

EtpP\u2013CDDP NLCs showed a uniformly spherical morphology (Figure 2<\/a>) with a size of 176.8\u00b14.9 nm and \"image\"<\/span>-potential of \u201331.9\u00b13.2 mV (Table 1<\/a>). The EE of NLCs was around 90% and that of LCs 3.3%\u20135.4%. The stability of NLCs was evaluated by changes in size, presented in Figure 3<\/a>. NLC formulations showed good stability during 90 days of storage in PBS at 2\u00b0C\u20138\u00b0C, while NLCs remained stable in the first 6 days (Figure 4<\/a>). 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.<\/p>\n\n\n\n
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Table 1<\/strong> Characterization of NLCs (means \u00b1 SD, n=8)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
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Figure 3<\/strong> The stability of NLCs evaluated in PBS at 2\u00b0C\u20138\u00b0C (A<\/strong>) and cell-culture medium (DMEM + 10% FBS) at 37\u00b0C (B<\/strong>) by changes in size with time.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
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Figure 4<\/strong> In vitro release of Etp (A<\/strong>) and CDDP (B<\/strong>) 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\u00b0C with a rotation speed of 100 rpm.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Cellular Uptake<\/h3>\n

Figure 5<\/a> illustrate the cellular uptake efficiency of EtpP\u2013CDDP NLCs, Etp\u2013CDDP NLCs and blank NLCs: 57.4%\u00b13.9%, 55.1%\u00b14.1% and 59.2%\u00b13.5% 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 al28<\/a>,37<\/a><\/sup>\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 5<\/strong> Cellular uptake efficiency of EtpP\u2013CDDP NLCs, Etpp\u2013CDDP NLCs, and blank NLCs. Uptake of NLCs was visualized using inversion fluorescence microscopy and quantified by fluorescence-activated cell sorting.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Cytotoxicity and Synergistic Effect<\/h3>\n

Blank NLCs did not change cell viability, while free drug and drug-loaded NLCs showed obvious cell-inhibition efficiency compared with the control (P<\/em><0.05) EtpP\u2013CDDP NLCs exhibited higher cytotoxicity than Etp\u2013CDDP NLCs, EtpP NLCs, and free EtpP\u2013CDDP on A549\/DDP cells (Figure 6A<\/a> and Supplementary Figure 1<\/a><\/u>, P<\/em><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<\/a>). The CI50<\/sub> values that illustrated the synergistic effect of the dual drug\u2013containing systems are presented in Figure 6D<\/a>, which shows that CI values were <1 with fraction of affected cells (Fa) of 20%\u201380% (0.2\u20130.8). Caspase 3 activity assays showed that caspase activity in A549\/DDP cells treated with EtpP\u2013CDDP NLCs increased significantly in comparison to Etp\u2013CDDP NLCs, EtpP NLCs, and free EtpP\u2013CDDP (P<\/em><0.05). This indicated that EtpP\u2013CDDP NLCs promoted caspase 3 activation in lung cancer cells (Figure 6E<\/a>).\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 6<\/strong> Cytotoxicity of drugs and drug-containing NLCs assessed by evaluating A549\/DDP (A<\/strong>), BEAS-2B (B<\/strong>), and HUVECs (C<\/strong>) viability using MTT assays. A549\/DDP and BEAS-2B cells (2\u00d7104<\/sup> cells\/well) were seeded in 96-well plates and allowed to grow for 24 h, then were treated with EtpP\u2013CDDP NLCs, Etpp\u2013CDDP NLCs, EtpP NLCs, CDDP NLCs, blank NLCs, free EtpP\u2013CDDP, free EtpP, and free CDDP at various drug concentrations. CI50<\/sub> values illustrated the synergistic effect of the dual drug\u2013containing systems (D<\/strong>). Caspase 3 activity assays were carried out according to the instructions of the manufacturer with a caspase 3 activity kit (E<\/strong>). *P<\/em><0.05.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

In Vivo Pharmacokinetics and Tissue Distribution<\/h3>\n

The blood concentration\u2013time profiles of EtpP\u2013CDDP NLCs, Etp\u2013CDDP NLCs, and free EtpP\u2013CDDP are presented in Figure 7<\/a>. EtpP\u2013CDDP NLCs and Etp\u2013CDDP NLCs exhibited more sustained plasma retention effects than the rapidly decreasing concentration of free EtpP\u2013CDDP. Tissue biodistribution of drugs is summarized in Figure 8<\/a>. At both 1 and 48 h after intravenous injection, EtpP\u2013CDDP NLCs showed higher drug distribution in tumors than Etp\u2013CDDP NLCs and free EtpP\u2013CDDP (Supplementary Figures 2<\/a><\/u> and 3<\/a><\/u>, P<\/em><0.05). Etp\u2013CDDP NLCs exhibited higher tumor drug accumulation than free EtpP\u2013CDDP (P<\/em><0.05). Tumor accumulation of free EtpP\u2013CDDP decreased rapidly after 48 h, while NLC formulations remained at high levels at 48 h.\n<\/p>\n\n\n\n
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Figure 7<\/strong> Etp (A<\/strong>) and CDDP (B<\/strong>) blood concentration\u2013time profiles of EtpP\u2013CDDP NLCs, Etpp\u2013CDDP NLCs, and free EtpP\u2013CDDP. Mixtures were vortexed and centrifuged (15,000 rpm, 10 min), and supernatants and plasma concentration of drugs were determined.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 8<\/strong> Tissue Etp (A<\/strong>) and CDDP (B<\/strong>) biodistribution of EtpP\u2013CDDP NLCs, Etpp\u2013CDDP NLCs, and free EtpP\u2013CDDP. 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<\/em><0.05.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

In Vivo Antitumor Ability<\/h3>\n

In vivo antitumor ability of drug-loaded NLCs and free drugs were investigated on lung cancer\u2013bearing mice (Figure 9<\/a>). Drug-loaded NLC groups showed remarkably higher tumor inhibition compared to free drugs (Figure 9A<\/a> and Supplementary Figure 4<\/a><\/u>, P<\/em><0.05). Dual drug\u2013loaded EtpP\u2013CDDP NLCs exhibited higher inhibit rates than single drug\u2013loaded NLCs (P<\/em><0.05). Most importantly, EtpP\u2013CDDP NLCs illustrated enhanced antitumor efficacy than non-prodrug\u2013containing Etp\u2013CDDP NLCs (P<\/em><0.05). Figure 9B<\/a> shows that drug-containing NLCs did not cause changes in body weight, while free drugs caused a decrease in weight (Supplementary Figure 5<\/a><\/u>, P<\/em><0.05). An increase in Cre was observed in the free-drug groups (Figure 9C<\/a>), while the Alt and WBC values of NLCs and free drugs were not obviously changed (Figure 9A and B<\/a>).\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 9<\/strong> In vivo antitumor activity (A<\/strong>), body-weight changes (B<\/strong>), WBC (C<\/strong>), Alt (D<\/strong>), and Cre (E<\/strong>) in lung cancer\u2013bearing mice. Tumor sizes were measured using calipers before every injection and tumor volume calculated by long axis \u00d7 (short axis)2<\/sup>\/2. Body-weight changes were monitored every 3 days to evaluate systemic toxicity. *P<\/em><0.05.<\/p>\n

Note<\/strong>: Data presented as means \u00b1 SD, n=6.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion<\/h2>\n

At the beginning of this study, an Etp prodrug was synthesized. Under controlled conditions (DMAP and CH2<\/sub>Cl2<\/sub>), the alcohol groups of the Etp glucose moiety did not react, and only the \u2013OH phenol group was coupled with Etp.26<\/a><\/sup> After that, dual drug\u2013loaded NLCs were prepared. NLCs have been reported to be nanosized with narrow size distribution and high drug EE.38<\/a><\/sup> The size of the prepared NLCs was around 170 nm, which proved to be efficiently internalized into cells.39<\/a><\/sup> The EE of NLCs was about 90%, which promised good loading ability of the systems.40<\/a><\/sup><\/p>\n

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<\/a><\/sup> 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<\/a><\/sup> Cellular uptake of nanoparticles can be quantitatively detected by the fluorescence method.39<\/a><\/sup> Cellular uptake efficiency of NLCs was >50%, which may be recognized as relatively high.33<\/a>,37<\/a><\/sup><\/p>\n

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<\/a><\/sup> EtpP\u2013CDDP NLCs exhibited higher cytotoxicity than Etp\u2013CDDP NLCs, EtpP NLCs and free EtpP\u2013CDDP 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<\/a><\/sup> 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<\/a><\/sup> 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<\/a><\/sup> The CI50<\/sub> values of the systems tested in this research were <1, which illustrated the synergistic effect of the dual drug\u2013containing systems.<\/p>\n

In vivo pharmacokinetic and tissue-distribution studies were carried out on lung cancer\u2013bearing mice. Higher accumulation in tumor tissue may contribute to the passive targeting ability of nanoparticles through the enhanced permeability-and-retention (EPR) effect.46<\/a><\/sup> 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<\/a><\/sup> EtpP\u2013CDDP NLCs showed the most significant antitumor efficiency (P<\/em>=0.0186), better than non-prodrug\u2013containing Etp\u2013CDDP NLCs. This result is in accordance with the findings of Wang et al that prodrug-based systems can improve lung cancer chemotherapy.47<\/a><\/sup> 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<\/a><\/sup><\/p>\n

Conclusion<\/h2>\n

An Etp prodrug was synthesized and EtpP\u2013CDDP NLCs prepared. EtpP\u2013CDDP 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.<\/p>\n<\/div>\n

\n

Disclosure<\/h2>\n

The authors report no conflicts of interest in relation to this work.<\/p>\n

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