- Email: [email protected]
Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery
Accepted Manuscript Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery Qing Xia, Longbing Ling, Muhammad Ismail, Yawei Du, Wei He, Wenya Zhou, Chen Yao, Xinsong Li PII:
S1773-2247(19)30022-X
DOI:
https://doi.org/10.1016/j.jddst.2019.03.010
Reference:
JDDST 973
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 5 January 2019 Revised Date:
4 March 2019
Accepted Date: 10 March 2019
Please cite this article as: Q. Xia, L. Ling, M. Ismail, Y. Du, W. He, W. Zhou, C. Yao, X. Li, Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.03.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract The self-assembly of artesunate-phospholipid (di-ART-PC) based liposomes for
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combinatorial PTX loading.
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Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery
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Qing Xia, Longbing Ling, Muhammad Ismail, Yawei Du, Wei He, Wenya Zhou, Chen Yao, Xinsong Li* School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, P R China *Contact Information of the Corresponding Author Xinsong Li PhD; Email: [email protected]
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Abstract Codelivery of two or multiple chemotherapeutics for enhanced anticancer efficacy is a commonly used strategy in cancer treatment, but the essential antagonistic performance of the combined drugs has limited their utilization. In this study, a novel active dual functional liposomal formulation based on the dimeric artesunate glycerophosphorylcholine (di-ART-PC) conjugate was developed for synergistical delivery of the established paclitaxel (PTX). The optimized PTX/di-ART-PC liposomes were demonstrated with high drug loading content (10.52%) and loading efficiency (83.9%) of PTX, featuring the reasonable size (174.6 nm) and well-defined spherical structure. In vitro release profiles revealed that PTX/di-ART-PC liposomes have controlled release profile of PTX and ART at a weakly acidic environment. Moreover, the nanoscaled characteristics and dual functionality make the resultant 2-in-1 liposomes capably internalized into tumor cells and thereby, synergistically enhanced antitumor response, as confirmed by in vitro experiments. In particular, the liposomes displayed more efficacious antitumor effects compared with free drug cocktail solution, at which the combination index (CI) of PTX/di-ART-PC liposomes for MCF-7, HepG-2 and A549 cells were 0.753, 0.724 and 0.606 µg/mL, respectively. These results suggest that di-ART-PC liposomes will be a promising candidate for synergistic delivery of chemotherapeutics in 2-in-1 system to improve cancer treatment integrating efficacy. Keywords: Artesunate; Liposome; Paclitaxel; Synergistic delivery; Anticancer efficacy. 1. Introduction Chemotherapy remains an aggressive strategy for clinical treatment of cancers, a major threat to human health and life. Despite this, its utilization is strongly restricted by the unselective and short-lived therapeutic effect. Previous researches demonstrated that these failures are mainly ascribed to the unacceptable degree of side
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toxicity and heterogeneity of tumor cells, which possibly leads to the drug resistance [1,2]. In the present, combination therapy is offering the versatile platform to address the above-mentioned issues for more effective suppression of tumor cell growth, invasion and metastasis. Hardly could the antitumor agents reach the maximum benefits in cancer therapy useless they were accompanied with the additional drugs in a different targeted manner against cancer cells. For instance, the clinical case of the combination of PTX and carboplatin or vinorelbine, which were applied to treat non-small cell lung cancer (NSCLC), respectively, exhibited the significant therapeutic efficacy than the single-agent therapy [3-5]. However, the conventional combination therapy is crucially needed to further enhancement. It was identified that the distinctive pharmacokinetics of individual agents within a traditional cocktail may cause undesirable noncoordinated distribution and adverse effects during systemic administration, which thereby present the difficulty for clinical speculating in vivo anticancer efficacy based on in vitro synergistic cytotoxicity. A more efficacious combination approach that is able to coordinate the pharmacokinetics of codelivered antitumor drugs is highly demanded for maximum the combined effects without systemic toxicity [6,7]. Recently, nanotechnology has been developed as an innovative combination strategy by codelivery of multiple therapeutic agents into a single nanocarrier [8]. Nanoparticles that are regarded as promising drug delivery systems for cancer therapy are principally depended on the alternative pharmacokinetics of extended circulation in blood, targetly accumulated in tumor sites and reduced side toxicity via enhanced permeation and retention (EPR) effect [9,10]. More highlightly, the nanoscaled characterics of particles could provide the potential to coordinate the pharmacokinetic behavior of formulated drugs with maximizing anticancer activities and reducing side effects. Over the past decades, several taxane nanoformulations were developed, including albumin nanoparticles, liposomes, micelles, nanoemulsions, solid lipid nanoparticles, and nanocapsules [11,12]. The albumin-bound PTX (Abraxane®) could provide the potential to coordinate the pharmacokinetic behavior of formulated drugs in combination with maximizing anticancer activities and reducing side effects [13]. Compared with other nano-delivery systems, liposomes exhibited excellent capability of synchronous codelivery of various drugs with different hydrophilicity/hydrophobicity to tumor sites [14-16]. However, conventional liposomal formulations always suffer from poor stability and low loading efficiency, extremely limited their therapeutic outcomes in clinic. As reported previously, the maximal loading content of clinical PTX liposomal formulations including Lipusu® (Luye Pharma Group, China), EndoTAG®-1 (Medigene) and LEP-ETU (NeoPharm) is less than 4 mol%. The lipids assembled into liposomes are generally inert in nature without therapeutic effects and the application of extra excipient materials in
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liposomes may raise cost burden and risk the safety concerns [17-19]. Thus, a stable liposomal formulation with high drug loading and biological activity is essential for combinatorial drug delivery. To address these limitations, one fascinating approach is the construction of dual functional nanocarriers that enable themselves self-assembly with therapeutic effects and further can synergistically enhance the antitumor activity by codelivery of incorporated drugs [20-23]. A representative example of dual functional noncarrier is d-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) by esterification of hydrophobic Vitamin E succinate and hydrophilic PEG1000 segment. The amphiphilic TPGS forms core-shell spherical nanostructure in aqueous medium, which can synchronously deliver hydrophobic agents with its own Vitamin E to tumor sites. Previous studies have established the synergistic efficacy of TPGS-based delivery system in a couple of in vitro and in vivo evaluations [24-27]. Moreover, drug-phospholipid conjugate based nanocarrier systems have been explored for the delivery of various chemotherapeutics [28,29]. Pedersen et al. [30] developed a novel active nanocarrier by conjugating chlorambucil drug with phosphatidylcholine, which assembled into unilamellar vesicles and displayed considerable cytotoxic potential against cancer cell lines. Prostaglandin-phospholipid conjugate was further synthesized and assembled forming vesicular structures in aqueous buffer with enhance cytotoxicities against HT-29 and Colo205 cells. Similarly, retinoid phospholipid conjugate-based liposomes were developed in 2010, which released retinoids under hydrolysis and displayed inhibition against colon cancer cells [31,32]. Recently, our group demonstrated the concept of dual drug-tailed phospholipid, replacing the two tails of the fatty acid chains by hydrophobic drug molecules and assembling itself into liposomes. For example, a liposomal formulation derived from dual chlorambucil-tailed phospholipid (DCTP) was developed to delivery chlorambucil, which exhibited significant antitumor efficacy with reduced side effects, highlighting their potential applications in combinatorial therapy [33]. Artesunate (ART), a naturally anti-malarial compound, shows anticancer activities against a variety of breast cancer, ovarian cancer and melanoma cancer cells [34]. Notably, it possesses the favorable selective property that is toxic to cancer cells but well safe towards normal cells, especially in case of combinational chemotherapy, which would benefit the biocompatibility after in vivo administration [35]. Based on this rational, we designed a novel active dual functional liposomal formulationbased on di-ART-PC conjugate with the aim to realize the precise codelivery of other payloads and synergistic prevention of tumor growth. PTX, as a first-line clinical anticancer drug, was passively loaded into the nanocarrier to formulate PTX/di-ART-PC liposomes with defined ratios by a typical dried thin-film technique. The physicochemical characterizations, stability and in vitro release behavior of
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2. Materials and Methods 2.1. Regents Artesunate (ART, purity ≥ 98%) was provided by Yuanye Bio-Technology Co., Ltd (Shanghai, China). Glycerylphosphorylcholine (GPC, purity ≥ 98%) was purchased from Innochem Technology Co., Ltd (Beijing, China). 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), N,N’-carbonyldiimidazole (CDI), tert-butyl diphenyl chlorosilane (TBDPSCl, purity ≥ 97%), tetra-butylammonium fluoride (TBAF) and dimethyl sulphoxide (DMSO) were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). ε-caprolactone, dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) and dichloromethane (DCM) were obtained from Juyou Scientific Equipment Co., Ltd (Nanjing, China). N,N’-dimethylformamide (DMF) and tetrahydrofuran (THF) were received from Sinopharm Chemical Reagent Co., Ltd (Nanjing, China). Unless otherwise stated, all the chemicals and reagents were of analytical grade. MCF-7 cells (human breast carcinoma), A549 cells (human lung carcinoma) and HepG-2 cells (human liver carcinoma) were gifted from the Chinese Academy of Sciences (Shanghai China). Secreted phospholipases A2 (sPLA2) was purchased from Mall Bio-Technology Co., Ltd (Nanjing, China). Fetal bovine serum (FBS), methyl tetrazolium (MTT), RPMI 1640 medium, cyanine (Cy5.5) and trypsin without EDTA were purchased from KeyGen Biotech Co., Ltd (Nanjing, China).
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2.2. Instruments Analytical high-performance liquid chromatography (HPLC, Agilent, CA) equipped with Agilent 1100 Infinity Quaternary HPLC System including a ZORBAX SB-C18 analytical column (4.6 × 150 mm) and a VWD UV-Vis detector. Mass spectrometry was carried out at Agilent 6540 instrument (MS, Agilent, CA). 1H NMR and 13C NMR spectra were performed on a Bruker DPX spectrometer at 500 MHz (Bruker, WI). 2.3. Synthesis of di-ART-PC conjugate Di-ART-PC prodrug was synthesized by covalent conjugation of artesunate (ART) to glycerophosphocholine (GPC) as shown in Scheme 1. The detailed steps are described below: Compound A: In the first step, ε-caprolactone (10 g, 0.087 mol) was added gradually
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to a solution of sodium hydroxide (NaOH, 3.5 g, 0.087 mol) in 100 mL water. After stirred at 25 °C for 12 h, the solution was extracted with ethyl acetate (EtOAc, 3 × 80 mL) and dried the solvent to get a white solid A (12.9 g, yield 98%). The chemical structure of compound A was checked via MS and 1H NMR as presented in supporting information (Figure S1 and S2). MS (m/z): calcd for C6H12O3, 132.08; found, 155.0 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 5.33 (s, 1-H, H/1), 3.67 (t, 2-H, H/2, J = 6.5 Hz), 2.37 (t, 2-H, H/6, J = 7.4 Hz), 1.68 (m, 2-H, H/3), 1.61 (m, 2-H, H/5), 1.43 (m, 2-H, H/4) ppm.
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Compound B: In the second step, to a solution of compound A (2 g, 15 mmol) and imidazole (1.02 g, 15 mmol) in anhydrous DMF (30 mL), TBDPSCl (4.12 g, 15 mmol) was added gradually and the reaction was stirred at 25 °C overnight. After diluting the resultant mixture with DCM (80 mL) and subsequently washing with deionized water (30 mL × 3), the solvents were first evaporated and dried by anhydrous Na2SO4, followed by purification via silica column chromatography using (EtOAc: hexane, 1:3) as an eluent provided compound B as a colorless viscous liquid (4.32 g, yield: 77%). The chemical structure of this step was confirmed by MS and 1H NMR as presented in supporting information (Figure S3 and S4). MS (m/z): calcd for C22H30O3Si, 370.20; found, 371.20 [M+H]+, 393.18 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 7.76 (d, 4-H, H/1,1’,5,5’, J = 6.5 Hz), 7.44 (t, 6-H, H/2,2’,3,3’,4,4’, J = 7.4 Hz), 3.75 (t, 2-H, H/9, J = 6.5 Hz), 2.41 (t, 2-H, H/13, J = 7.5 Hz), 1.71-1.65 (m, 2-H, H/10), 1.54-1.48 (m, 2-H, H/12), 1.34-1.29 (m, 2-H, H/11), 1.14 (s, 9-H, H/6,7,8) ppm.
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Compound C: In the third step, 0.87 g/5.4 mmol of CDI was mixed with a solution of compound B (2 g, 5.4 mmol) dissolved in DCM (15.00 mL) and stirred at room temperature for 4 h. Meanwhile, 0.69 g/2.7 mmol of GPC dissolved in anhydrous DMSO (20.00 mL) was stirred with DBU (0.39 g, 2.6 mmol) for 4 h at 35 °C. Then, the two solutions were combinedand nightlystirred at room temperature. The reaction progress was observed through TLC (20% methanol in CH2Cl2 as an eluent, UV light at 254 nm). Purification of the product was carried out by silica column chromatography using CH2Cl2/CH3OH (solvent A: CH2Cl2:CH3OH, 10:1; solvent B: CH2Cl2:CH3OH:H2O 65:25:4) as an eluent, provided C as a white solid (3.5 g, yield: 33%). The structure of compound C was confirmed by MS and 1H NMR as presented in supporting information (Figure S5 and S6). MS (m/z): calcd for C52H76NO10PSi2, 961.47; found, 962.47 [M+H]+, 984.45 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 7.69 (d, 8-H, H/3,3’,4,4’,6,6’,10,10’, J = 6.5 Hz), 7.41 (t, 12-H, H/1,1’,2,2’,5,5’, 7,7’,8,8’, 9,9’, J = 7.0 Hz), 5.24 (d, 2-H, H/20, J = 5.5 Hz), 4.44 (t, 2-H, H/22, J = 7.4 Hz), 4.16 (m, 1-H, H/19), 3.99 (t, 2-H, H/23, J = 7.5 Hz), 3.93 (t, 4-H, H/14,14’, J = 7.0 Hz), 3.72 (d, 1-H, H/21, J = 7.4 Hz), 3.29 (s, 9-H, H/24, 25,26), 2.33-2.26 (m, 4-H,
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Compound D: In the final step, the protection of hydroxyl (-OH) group in compound C by TBDPSCl agent was deprotected using TBAF solution. Briefly, TBAF (1 M, 2 mL) of was added to compound C solution (1 g, 1.0 mmol) in THF (30 mL). The reaction mixture was continuously stirred for 2 h at ambient temperature, while their completion was observed with TLC (solvent: 20% methanol in CH2Cl2, UV light at 254 nm). To this obtained product without further purification, ART (0.79 g, 2.78 mmol), EDC.HCl (0.53 g, 2.80 mmol) and DMAP (0.15 g, 1.22 mmol) were added in 30 mLof DMSO and over nightly stirred at 25 °C. The mixture was subjected to chromatographic purification, which first eluted with solvent A (CH2Cl2:CH3OH, 10:1) and then with solvent B (CH2Cl2:CH3OH:H2O, 65:25:4), afforded the white solid compound D (di-ART-PC, 0.31 g, yield: 25%). The structure of di-ART-PC was verified by MS, 1H NMR and 13C NMR. (0.31 g, yield: 25%). MS (m/z): calcd for C57H92NO24P, 1217.5; found, 1218.7 [M+H]+, 1240.7 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 5.79 (d, 2-H, H/10,10’, J = 9.8 Hz), 5.45 (s, 2-H, H/8,8’), 5.22 (d, 2-H, H/26, J = 5.5 Hz), 4.33 (t, 2-H, H/29, J = 10.5 Hz), 4.15 (m, 1-H, H/27), 4.09 (t, 4-H, H/20,20’, J = 6.0 Hz), 3.97 (t, 2-H, H/30, J = 6.5 Hz), 3.79 (d, 2-H, H/28, J = 7.4 Hz), 3.36 (s, 9-H, H/31,32,33), 2.78-2.50 (m, 10-H, H/13,13’,21,21’,22,22’), 2.46-2.26 (m, 8-H, H/18,18’,24,24’), 2.10-1.58 (m, 24-H, H/4,4’,5,5’,14,14’,15,15’,17,17’,23,23’), 1.44 (s, 6-H, H/7,7’), 1.40-1.02 (m, 6-H, H/2,2’,3,3’,11,11’), 0.98 (d, 6-H, H/1,1’, J = 5.7 Hz), 0.87 (d, 6-H, H/12,12’, J = 6.9 Hz) ppm. 13C NMR (500 MHz, CDCl3): δ 172.37, 171.62, 170.65, 103.91, 91.65, 90.98, 79.60, 70.21, 66.04, 64.08, 62.99, 62.45, 58.71, 54.02, 51.06, 44.72, 36.71, 35.73, 33.63, 33.33, 31.28, 28.69, 28.29, 27.66, 25.46, 24.88, 24.77, 24.04, 23.89, 21.45, 19.69, 11.52 ppm.
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2.4. Critical micelle concentration (CMC) of di-ART-PC conjugate Pyrene fluorescent probe-technique was used to determine the critical micelle concentration (CMC) of di-ART-PC conjugate on a HJYFL3-211-TCSPC spectrometer (HORIBAJobin Yvon, France). In detail, di-ART-PC conjugate was solubilized in distilled water and diluted a series of concentrations from 70 µg/mL to 5µg/mL, where, each tube contained a total of 1 mL solution. A pyrene solution having a concentration of 5×10-5 M in acetone was added into di-ART-PC solutions in different tubes. After equilibration at 25 °C for 24 h, the samples were subjected to HJYFL3-211-TCSPC spectrometer to record fluorescence spectra of each solution. The excitation wavelength of the fluorescence scan was 334 nmwith the excitation slit of 5.0 nm and the emission slit was set to 2.5 nm. The CMC value of di-ART-PC conjugate was determined from the intensity ratio of the first vibronic band (I1, λ1 is
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2.5. Characterization of di-ART-PC and PTX/di-ART-PC liposomes Both di-ART-PC and PTX/di-ART-PC liposomes were formulated by employing reverse-phase evaporation technique according to the previous studies [36-38]. For di-ART-PC liposomes, di-ART-PC conjugate (10 mg, 8.21 mmol) was solubilized in 30 mL dichloromethane, following the addition of PBS buffer (pH 7.4, 10 mL) and the resulted emulsion was ultrasonicated by Ymnl-150Y for 30 min. DCM was removed by evaporation, and the liposomes were rotated at 50 °C for 1 h in rotary evaporator. Finally, the di-ART-PC liposomes were homogenized by a 0.22 um filter membrane with a micro-extruder at 50 °C. For PTX/di-ART-PC liposomes, di-ART-PC and PTX in different ratios (Table 1) were dissolved in DCM (30 mL), and then added 10 mL of PBS buffer (pH 7.4). Afterward, Ymnl-150Y supersonic probe was applied to sonicate the obtained emulsion for 30 min, followed by evaporation at rotary evaporator. The residual solvent, un-loaded PTX and un-assembled di-ART-PC were removed by dialysis in PBS (pH 7.4, 200 mL) for 12 h to obtain a final suspension of the PTX/di-ART-PC liposomes. Additionally, the conventional ART and PTX-loaded liposomes composed of lecithin/Chol/DSPC-PEG (90:10:5 Mol% ratio) (combo-liposomes) were prepared as similar as the above procedure to compare their in vitro antitumor effects with PTX/di-ART-PC liposomes. Size and zeta-potential of the afforded liposomes were measured by DLS. The morphology of di-ART-PC liposomes was analyzed by using transmission electron microscopy (TEM). Samples were prepared by dropping 10 µL of liposomes solution on a 200-mesh carbon film copper grid, air-drying and negatively staining (phosphomolybdic acid, 2% w/v). Observation was carried out by JEM-2100 TEM (JEOL, Japan) at 200 kV. 2.6. In vitro release of ART and PTX The drug release profiles of PTX/di-ART-PC liposomes were estimated in simulated neutral (PBS, pH 7.4) and weakly acidic (PBS, pH 5.0) release mediums. For release experiment, PTX/di-ART-PC liposomal suspension (3 mL) was incubated in a dialysis bag (MWCO, 3500) in a tube containing release medium (30 mL) supplemented with 1% Tween 80 at 37 °C. The tubes were kept in Shaker Incubator LSI-100B under shaking condition (100 rpm). At predetermined time points, the release medium (1 mL) containing released drugs was taken and immediately substituted with an equivalent volume of fresh media. PTX and ART contents were analyzed by means of HPLC under eluting solvents of 80% MeCN in water (0.1%
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TFA). Furthermore, the release of di-ART-PC liposomes was performed with secretory phospholipase A2 (sPLA2) according to the reported literature [39,40]. The catalytic release was initiated by adding 2 µL of a 45 µM sPLA2 stock solution to 20 mL of 25 µM di-ART-PC liposomes at 37 °C equilibrated 20 min prior to addition of the enzyme. The release of ART was measured by HPLC with 20 µL of solution sample taken every ten minutes. All experiments were repeated thrice and the results were expressed as the mean ± standard deviation.
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2.7. In vitro cellular uptake The cellular uptake property of PTX/di-ART-PC liposomes labeled by Cy5.5 dye was analyzed via confocal laser scanning microscopy (CLSM) after incubation with MCF-7 cells. The Cy5.5-labelled di-ART-PC liposomes were formulated by reverse-phase evaporation method as described above under light protection conditions. MCF-7 cells were treated with Cy5.5-labelled di-ART-PC liposomes (5 µg Cy5.5/mL) for two different time periods (2 h and 4 h) at 4 °C or 37 °C, respectively. After washing the treated cells twicewith PBS and fixing by 4% paraformaldehyde, the cells were stained for 15 min with 4,6-diamidino-2-phenylindole (DAPI) at room
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temperature. The resultant slides were affixedon a glass slide for observation in a laser scanning confocal microscope (Leica TCS SP2, Leica Co., Wetzlar, Germany).
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2.8. In vitro cytotoxic activity The cell viability of PTX/di-ART-PC liposomes versus MCF-7, A549 and HepG-2 three different cancer cell lines was measured by MTT assay using free ART, free PTX, and conventional ART/PTX-loaded lecithin liposomes (combo-liposomes), as controls. In the experimental protocol, 100 µL of cell suspensions with the density of 7.5×103 cells per well was seeded in plates (96-well) and cultured at 37 °C overnight. Then, cells in each well were treated with various concentrations of PTX/di-ART-PC liposomes and control solutions, and incubated for additional 24 h under standard incubatory condition. In order to evaluate the cell viability after drug treatment, MTT solution (20 µL, 1 mg/mL,) was supplemented to all wells and continued incubation for 4 h. After the removal of medium, dimethyl sulfoxide (150 µL) was added to solubilize the developed crystals in the wells for 5 min. Finally, the microplate reader (Multiskan EX, Thermo Scientific, Waltham, MA) was applied to check the absorbance at 570 nm and to determine the cell viability as follows.
Cell viability (%)=
ODtreated − ODblank × 100 ODcontrol − ODblank
ODtreated represents the optical density (OD) from treatment groups and ODcontrol
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indicates the OD from growth medium, whereas, ODblank is the OD from MTT solution lacking cells, respectively. Combination index (CI) is commonly used to analyze the synergistic, antagonistic or additive effects of two drugs [41,42]. The ART and PTX drug combinatorial from PTX/di-ART-PC liposomes and combo-liposomes were used to calculate the CI values, where CI > 1 indicates antagonism; CI = 1 represents additive effect and CI < 1 designates synergistic effects. CI was calculated by the following formula: C1 C2 CI = + Cx1 Cx2 Whereas, C1 and C2 is the concentration of drug 1 and drug 2 to produce a certain effect in combination, while Cx1 and Cx2 represents the concentration of drug 1 and 2 generating identical effect alone [43-45]. GraphPad sigmoid curves (efficacy–dose–response relationships) were used to determine the degree of effect foreach drug for calculating CI [46-49]. 2.9. Cell apoptosis assay MCF-7 cells at a concentration of 1×105 cells per well were cultured in 12-well plates. Post-incubation for 24 h, the cells were treated with free ART, free PTX, ART/PTX mixture, combo-liposomes and PTX/di-ART-PC liposomes, respectively at
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an equal drug concentration of 5 µg/mL. Then, the cells were incubated for 24 h at 37 °C. After incubatory period, the cells were washed twice with PBS, followed by treatment with binding buffer and staining through Annexin-V and PI (propidiumiodide) under dark (10 min). Finally, the cells were examined for apoptosis
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2.10. Hemolytic toxicity The hemolytic toxicity of di-ART-PC liposomes (both blank and PTX-loaded) was conducted by incubating with human red blood cells (RBCs) in order to check their blood compatibility. Briefly, 10 mL of fresh whole blood from healthy volunteers was centrifuged on 1500 rpm at 4 °C for 10 min to take away the supernatant. The precipitated red blood cells were washed thrice with saline till the supernatant was clear. The obtained red blood cells were mixed with normal saline to prepare a 2% erythrocyte suspension (2 mL of RBCs and normal saline to 100 mL). Then, 25 µL aliquots of erythrocyte suspension were exposed to blank di-ART-PC liposomes at concentrations of 5, 25 and 50 µg/mL, and to PTX/di-ART-PC liposomes at PTX concentrations of 1, 5 and 10 µg/mL. The samples were incubated in a shaker for 4 h at 37 °C and then centrifuged at 1500 rpm for 15 minutes. Supernatants were collected and the absorbance of hemoglobin was measured at 545 nm through UV-Vis
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spectrometer. The selected normal saline and deionized water causing zero hundred percent hemolysis were used as negative and positive controls, respectively. The hemolysis ratio lower than 5% was interpreted safe. The % hemolysis rate was determined by the following formula: Aexp − Aneg Hemolysis rate % = × 100 Apos − Aneg where, Aexp is the absorbance of experimental group, where, Apos and Aneg are absorbance of positive and negative controls, respectively.
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3. Results and Discussion 3.1. Synthesis of di-ART-PC conjugate The di-ART-PC conjugate was synthesized by the covalent conjugation of ART with PC under CDI/DBU catalytic system as elucidated in Scheme 1. The high purity (96%) di-ART-PC prodrug with an overall yeild of 25% was obtained as indicated in Figure 1A (inset). The MS and 1H NMR spectra of the intermediates (A-C) are presented in supporting information (Figure S1-S6). The MS data of di-ART-PC conjugate shows the molecular iron peaks at 1218.7 (m/z, [M+H]+) and 1240.7 (m/z, [M+Na]+), that are in agreement with the calculated value (C57H92NO24P 1217.7, Figure 1A). 1H NMR spectrum (Figure 1B) of the di-ART-PC conjugate displayed signals from methyl protons of artesunate at 1.44 ppm, 0.98 ppm and 0.87 ppm, along
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with a prominent typical singlet at δ 3.36 ppm, that was attributed to the methyl protons of -N+ (CH3)3 group form GPC group. This result strongly supports the couple of ART with GPC. Moreover, the existed distinctive carbon signal at 54.02 ppm is attributed to the -N+ (CH3)3 head-group in 13C NMR spectrum of di-ART-PC (Figure
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1C). All these experimental results clearly established the successful synthesis of di-ART-PC prodrug.
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Scheme 1. Synthetic procedure of di-ART-PC conjugate. Reagents and conditions: (a) NaOH/H2O, RT, 12 h, 98%; (b) TBDPSCl, imidazole, DMF, RT, 12 h, 77%; (c) L -α-glycerophosphocholine, CDI, DBU, CH2Cl2, DMSO, RT, 12 h, 33%; (d) TBAF, THF, RT, 4 h, 96%; (e) ART, EDC.HCl, DMAP, THF/DMSO, RT, 12 h, 25%.
Scheme 2. Illustration of the self-assembly of di-ART-PC into liposomes for PTX loading.
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ACCEPTED MANUSCRIPT Figure 1. Characterization of di-LA-PC conjugate: (A) Mass spectrum, (inset) HPLC profile of di-ART-PC eluted with 80% MeCN in water (0.1% TFA in 18 min at 10.237 min; (B) 1H NMR (500 MHz, CDCl3) and (C) 13C NMR (500 MHz, CDCl3) spectra.
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3.2. Critical micelle concentration (CMC) of di-ART-PC conjugate Self-assembly ability of the di-ART-PC conjugate was verified by its critical micelle concentration (CMC), determined by pyrene fluorescent probe method. The existence of hydrophobic ART and hydrophilic GPC moieties facilitated the rapid assembly of di-ART-PC conjugate in aqueous medium. The intensity ratio (I3/I1) was recorded as a function of di-ART-PC concentration (Figure 2). Due to high sensitivity of pyrene to the surrounding polar medium, it readily showed a sharp shift in I3/I1 (emission intensity ratio) and hence the CMC value could be determined. Under lower di-ART-PC concentration, no substantial change in I3/I1 value was observed but upon the enhancement of conjugate concentration, the ratio of I3/I1 rapidly augmented
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which could be attributed to the deposition of pyrene in hydrophobic atmosphere. Based on the sharp change in the curve, CMC value was calculated as 22 µg/mL,
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highlighting the successful self-assembly of di-ART-PC conjugate in aqueous media.
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Figure 2. Relationship between the fluorescent intensity ratio (I3/I1) and di-ART-PC concentration dispersed in water. The CMC value is about 22 µg/mL. 3.3. Characterization of di-ART-PC liposomes The developed amphiphilic di-ART-PC conjugate having two ART moieties conjugated with phospholipid demonstrated proficient self-assembly ability in aqueous environment, resulted into di-ART-PC liposomes. The size and morphology of the liposomes were investigated by DLS and TEM. As illustrated in Supporting information (Figure S7), the blank di-ART-PC liposomes have small size (163.05 nm) and a narrow distribution (PDI: 0.283) along with negatively charged surface (zeta potential of -23.37 mV) in water, indicating their promising stability. In addition,
ACCEPTED MANUSCRIPT TEM images revealed the spherical nanostructure of the di-ART-PC conjugate assembled liposomes with a diameter of about 100 nm, which is close to that determined by DLS. Di-ART-PC conjugate and PTX at different ratios were used to attain optimized
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liposomal formulation (Table 1). As shown in Table 1, the di-ART-PC conjugate to PTX in the ratio of 10:2 (mole ratio) showed higher DLE (83.9%) and a favorable DLC (10.52%) compared to the already reported liposomes [50,51]. In addition, the liposomes loaded with different concentrations of PTX were incubated in PBS (pH 7.4) solution for 1-10 days under dialysis bag (MWCO, 3500), while their stability and drug encapsulation rate were determined as a function of time. As shown in Figure S8, the liposomes developed form the initial feed molar ratios of conjugate to PTX (10:0.5, 10:1, 10:2) showed better stability, where the drug loaded rate changes were less than 10%. Due to the optimal particle size, stability, and drug loading efficiency, the drug to
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carrier ratio of 2:10 was selected to prepare optimized PTX/di-ART-PC liposomes and used in the following experiments. The average size of the optimized PTX/di-ART-PC liposomes slightly increased to 174.60 nm (Figure 3), versus blank di-ART-PC liposomes as described above. This may be due to the fact that the drug PTX is loaded into the liposome and the inert space of these liposomes is increased. Besides, the storage stability of PTX/di-ART-PC liposomes under simulated physiological
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conditions (PBS pH 7.4) was investigated by DLS at 4 °C (Figure S9). It was found that the liposomes were stable without significant size variation even after 7 days
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incubation. Overall, the di-ART-PC conjugate has efficient self-assembly ability benefiting from their amphiphilicity, as well as demonstrated promising loading efficiency of other anticancer agents.
Figure 3. Characterization of PTX-loaded di-ART-PC liposomes: (A) size and zeta-potential measured by DLS, and (B) TEM image.
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di-ART-PC:PTX
b
PDI
Zeta-potential
c
DLC %
d
DLE %
10:0.5
167.05±3.58
0.261
-21.55
2.83
83.1
10:1
169.25±3.64
0.256
-18.11
5.43
82.0
10:2
174.60±4.21
0.224
-17.30
10.52
83.9
10:5
180.35±5.03
0.228
-15.84
19.31
68.3
b
Data are given mean ± standard deviation (n=3); PDI: Polydispersity index; cDLC:
Drugloading capacity; dDLE: Drug loading efficiency.
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a
Size
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3.4. In vitro release study In vitro release of ART and PTX from PTX/di-ART-PC liposomes was investigated by dialysis (MWCO 3500) under two different simulated pH conditions (PBS pH 7.4 or pH 5.0). As shown in Figure 5, no significant ART and PTX release
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(less than 20%) was observed after a 72 hour incubation in PBS pH 7.4, which demonstrates the stable liposomal nanostructure of di-ART-PC liposomes as well as stable encapsulation of PTX within the liposomes. This behavior of the liposomes is desirable for the good stability under biological environment. Under weakly acidic environment (PBS, pH 5.0), rapid release rate of both PTX and ART were found during the first 24 h and the overall sustained release of 74% and 63.5%, respectively
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were reached over the next 48 h. It is supposed that the PTX/di-ART-PC liposomes dissociate and subject to acidic hydrolysis to sustainably release parent drug ART. As shown in Figure S10B, significant ART peak at 2.783 min was observed along with a peak at 7.431 min of ART-lysophospholipid. After 2 h incubation,
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appropriate 80% of ART was released from di-ART-PC liposomes (Figure S10C). The results clearly demonstrated that di-ART-PC liposomes were effectively degraded to
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release free ART in the presence of catalytic sPLA2. Conclusively, these PTX-loaded di-ART-PC liposomes is relatively stable without premature drug release under neutral condition, but could rapidly release antitumor agents in an acidic or lipases environment, leading to killing of the tumor cells effectively.
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Figure 5. In vitro release of ART and PTX from PTX/di-ART-PC liposomes under different simulated pH conditions for 72 h.
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3.5. Cellular uptake of di-ART-PC liposomes The cellular uptake behavior of di-ART-PC liposomes in MCF-7 cells was investigated by confocal laser scanning microscopy. For ease of observation, near-infrared fluorescence (NIRF) Cy5.5 was used to label di-ART-PC liposomes, while the cellular nuclei were stained by DAPI with blue fluorescence. As shown in Figure 6, the internalization of di-ART-PC liposomes into the MCF-7 cells can be clearly observed. The nucleus appears blue, as opposed to the cytoplasmic region (red), demonstrating that the di-ART-PC liposomes can enter into cytoplasm. These observations indicate that the di-ART-PC liposomes (red stained) are predominantly distributed throughout the cytoplasm. It has been reported that the mechanism of particle uptake is phagocytosis, liquid phase pinocytosis or receptor-mediated endocytosis. The main internalization mechanism of 100 nm particles is endocytosis, supporting the distribution of the liposomes into the cytoplasm as well as in the nucleus. Cy5.5-labelled di-ART-PC liposomes showed more definitive uptake during the incubation for 2 hours at 37 °C compared to 4 °C, and cellular uptake increased within 4 hours. It is therefore suggested that di-ART-PC liposomes encapsulated with PTX could enter into the cells and could effectively accumulate in the nucleus to improve the therapeutic effects in cancer therapy.
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Figure 6. Confocal laser scanning microscope analysis of Cy5.5-labelled di-ART-PC liposomes, showing their uptake into MCF-7 cells at 37 °C or 4 °C for 2 h and 4 h. Cell nuclei were stained by DAPI (blue). Scale bar: 25 µm.
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3.6. In vitro cytotoxicity of PTX/di-ART-PC liposomes Cytotoxicity is a major concern when developing delivery systems for anticancer drugs. Therefore, the in vitro cytotoxicity of PTX/di-ART-PC liposomes was determined by standard MTT assay and compared with different free drug and drug combinations against MCF-7, HepG-2 and A549 cells. The cytotoxic activity of PTX/di-ART-PC liposomes enhanced with the increase in drug concentration. Importantly, the PTX/di-ART-PC liposomes demonstrated significant anti-tumor effects that are higher than free ART, free PTX and conventional ART/PTX-loaded lecithin liposomes (combo-liposomes) (Figure 7). These findings suggested that PTX/di-ART-PC liposomes have better anticancer effects. As indicated in Table 2, the half maximal inhibitory concentration (IC50) values of PTX/di-ART-PC liposome versus MCF-7, HepG-2and A549 cancer cells were determined to be 2.9 ± 0.11 µg/mL, 4.3 ± 0.22 µg/mL, 3.0 ±0.14 µg/mL, respectively. These results were far significantly lower than control groups (Table 2). In this study, the combination index CI values of the PTX/di-ART-PC liposomes for MCF-7, HepG-2 and A549 cells were 0.753 µg/mL, 0.724 µg/mL and 0.606 µg/mL,
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combinatorial antitumor drug delivery. Based on the above data, PTX/di-ART-PC liposomes effectively delivered both antitumor agents to cancer carcinomas, and produced improvement in in vitro cytotoxic effects synergistically.
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Figure 7. Cell viability of free ART, free PTX, conventional combo-liposomes, and PTX/di-ART-PC liposomes. Error bar represent mean ± standard deviation (n = 4).
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Table 2. IC50 Values of ART, PTX, combo-liposomes, and PTX/di-ART-PC liposomes against MCF-7, Hepg2 and A549 cancer cells after 24 h incubation. Error bar represent mean ± standard deviation (n = 3). IC50 (µg/mL)
Formulation
MCF-7
HepG-2
A549
ART
34.6± 0.13
57.2± 0.19
37.5± 0.15
9.3± 0.21 4.5± 0.41
13.4± 0.31 6.1± 0.26
11.8± 0.27 4.1± 0.33
2.9± 0.11
4.3± 0.22
3.0± 0.14
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Table 3. Combination index values of combo-liposomes and PTX/di-ART-PC liposomes in three cancer cell lines. CI (µg/mL) Cells MCF-7 HepG-2 A549
Drug combination Combo-liposomes
PTX/di-ART-PC
0.915
0.753
0.924 0.909
0.724 0.606
3.7. Cell apoptosis assay Apoptotic assay was applied to evaluate the killing mechanism of MCF-7 cells treated with free ART, free PTX, conventional combo-liposomes and PTX/di-ART-PC
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control. The apoptotic results were shown in Figure 8. Among the treatment groups, PTX/di-ART-PC liposomes exhibited higher percentage of cells in early and late apoptosis compared to control groups at the same equivalent concentration. This apoptotic result is consistent with the MTT data. More specifically, the percentages of early apoptotic cells referred as Q4 (annexin positive/PI negative) for ART, PTX, conventional combo-liposomes and PTX/di-ART-PC liposomes were 2.38 ± 2.19%, 2.70 ± 2.56%, 4.33 ± 3.01% and 4.02 ± 2.89%, whereas those of late apoptotic cells (referred as Q2, annexinand PI double positive) were 6.61 ± 3.32%, 9.77 ± 2.19%, 14.0 ± 2.41% and 18.1 ± 2.57%, respectively (Figure 9). These results clearly demonstrated that PTX/di-ART-PC liposomes could increase the PTX-induced
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apoptosis upon their entrapment into active di-ART-PC nanocarrier compared to the plain PTX at equivalent concentration.
Figure 8. Flow cytometry induced apoptotic analysis of MCF-7 cells by ART, PTX, conventional combo-liposomes and PTX/di-ART-PC liposomes, at the same concentration (5 µg/mL) for 24 h. Nontreated cells were exploited as control. In each flow cytometry plot, Q1 refers to necrosis, Q2 late apoptosis, Q3 early apoptosis and Q4 viable cell.
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Figure 9. Quantitative analysis of MCF-7 cells apoptosis. Data are expressed as mean ± SD. 3.8. Hemolytic toxicity An important criterion for in vivo application is the blood compatibility of a nanocarrier after administration that is assessed by hemolysis study. Thus, the
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hemocompatibility of unloaded di-ART-PC and PTX/di-ART-PC was evaluated at different concentrations by incubating with human normal red blood cells (RBCs) as an in vitro toxicity model. The changes in hemolysis rate as a function of concentration induced by the treatment groups were presented in Figure 10. After incubation for 4 hours at 37 °C, the unloaded and PTX-loaded di-ART-PC liposomes were found to be non-hemolytic (<5%). The data significantly indicated that the di-ART-PC and PTX/di-ART-PC nanocarrier systems have good biocompatibility for in vivo administration.
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Figure 10. Hemolysis of unloaded di-ART-PC and PTX/di-ART-PC after 4 h incubation with blood erythrocytes at 37 °C. Each value represents the mean ± SD of three experiments.
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4. Conclusion In summary, a successful formulation based on active di-ART-PC liposomal nanocarrier platform was developed for PTX delivery for cancer treatment. Hydrophobic PTX was effectively encapsulated into the core of liposome with an impressive drug loading capacity and significant stability. The lower and uniform distributed size (<200 nm) of the PTX/di-ART-PC liposome demonstrated efficient internalization into tumor cells. In vitro cytotoxicity assay and apoptosis assays confirmed the effectiveness of the liposomes that has higher tumor inhibition and a good synergistic therapeutic effect (CI value < 1). All these results suggested that PTX/di-ART-PC liposomes may be a promising combination of anti-cancer regimens that could effectively improve the therapeutic effects. Moreover, the active dual functional di-ART-PC assembled liposomes can be a great promising candidate to synergistically delivery of anticancer therapeutics, thus offering a versatile platform in combinatorial nanomedicines. Acknowledgement The current work was funded by the Major National Science and Technology Program of China for Innovative Drug (2017ZX09101002-001-004), the National Natural Science Foundation of China (No. 51373034), the Department of Science & Technology of Jiangsu Province, China (Projects BA2013037, BY2015070-11) and the Priority Academic Program Development of Jiangsu Higher Education
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Conflicts of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery”.
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S1773-2247(19)30022-X
DOI:
https://doi.org/10.1016/j.jddst.2019.03.010
Reference:
JDDST 973
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 5 January 2019 Revised Date:
4 March 2019
Accepted Date: 10 March 2019
Please cite this article as: Q. Xia, L. Ling, M. Ismail, Y. Du, W. He, W. Zhou, C. Yao, X. Li, Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.03.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract The self-assembly of artesunate-phospholipid (di-ART-PC) based liposomes for
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combinatorial PTX loading.
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Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery
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Qing Xia, Longbing Ling, Muhammad Ismail, Yawei Du, Wei He, Wenya Zhou, Chen Yao, Xinsong Li* School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, P R China *Contact Information of the Corresponding Author Xinsong Li PhD; Email: [email protected]
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Abstract Codelivery of two or multiple chemotherapeutics for enhanced anticancer efficacy is a commonly used strategy in cancer treatment, but the essential antagonistic performance of the combined drugs has limited their utilization. In this study, a novel active dual functional liposomal formulation based on the dimeric artesunate glycerophosphorylcholine (di-ART-PC) conjugate was developed for synergistical delivery of the established paclitaxel (PTX). The optimized PTX/di-ART-PC liposomes were demonstrated with high drug loading content (10.52%) and loading efficiency (83.9%) of PTX, featuring the reasonable size (174.6 nm) and well-defined spherical structure. In vitro release profiles revealed that PTX/di-ART-PC liposomes have controlled release profile of PTX and ART at a weakly acidic environment. Moreover, the nanoscaled characteristics and dual functionality make the resultant 2-in-1 liposomes capably internalized into tumor cells and thereby, synergistically enhanced antitumor response, as confirmed by in vitro experiments. In particular, the liposomes displayed more efficacious antitumor effects compared with free drug cocktail solution, at which the combination index (CI) of PTX/di-ART-PC liposomes for MCF-7, HepG-2 and A549 cells were 0.753, 0.724 and 0.606 µg/mL, respectively. These results suggest that di-ART-PC liposomes will be a promising candidate for synergistic delivery of chemotherapeutics in 2-in-1 system to improve cancer treatment integrating efficacy. Keywords: Artesunate; Liposome; Paclitaxel; Synergistic delivery; Anticancer efficacy. 1. Introduction Chemotherapy remains an aggressive strategy for clinical treatment of cancers, a major threat to human health and life. Despite this, its utilization is strongly restricted by the unselective and short-lived therapeutic effect. Previous researches demonstrated that these failures are mainly ascribed to the unacceptable degree of side
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toxicity and heterogeneity of tumor cells, which possibly leads to the drug resistance [1,2]. In the present, combination therapy is offering the versatile platform to address the above-mentioned issues for more effective suppression of tumor cell growth, invasion and metastasis. Hardly could the antitumor agents reach the maximum benefits in cancer therapy useless they were accompanied with the additional drugs in a different targeted manner against cancer cells. For instance, the clinical case of the combination of PTX and carboplatin or vinorelbine, which were applied to treat non-small cell lung cancer (NSCLC), respectively, exhibited the significant therapeutic efficacy than the single-agent therapy [3-5]. However, the conventional combination therapy is crucially needed to further enhancement. It was identified that the distinctive pharmacokinetics of individual agents within a traditional cocktail may cause undesirable noncoordinated distribution and adverse effects during systemic administration, which thereby present the difficulty for clinical speculating in vivo anticancer efficacy based on in vitro synergistic cytotoxicity. A more efficacious combination approach that is able to coordinate the pharmacokinetics of codelivered antitumor drugs is highly demanded for maximum the combined effects without systemic toxicity [6,7]. Recently, nanotechnology has been developed as an innovative combination strategy by codelivery of multiple therapeutic agents into a single nanocarrier [8]. Nanoparticles that are regarded as promising drug delivery systems for cancer therapy are principally depended on the alternative pharmacokinetics of extended circulation in blood, targetly accumulated in tumor sites and reduced side toxicity via enhanced permeation and retention (EPR) effect [9,10]. More highlightly, the nanoscaled characterics of particles could provide the potential to coordinate the pharmacokinetic behavior of formulated drugs with maximizing anticancer activities and reducing side effects. Over the past decades, several taxane nanoformulations were developed, including albumin nanoparticles, liposomes, micelles, nanoemulsions, solid lipid nanoparticles, and nanocapsules [11,12]. The albumin-bound PTX (Abraxane®) could provide the potential to coordinate the pharmacokinetic behavior of formulated drugs in combination with maximizing anticancer activities and reducing side effects [13]. Compared with other nano-delivery systems, liposomes exhibited excellent capability of synchronous codelivery of various drugs with different hydrophilicity/hydrophobicity to tumor sites [14-16]. However, conventional liposomal formulations always suffer from poor stability and low loading efficiency, extremely limited their therapeutic outcomes in clinic. As reported previously, the maximal loading content of clinical PTX liposomal formulations including Lipusu® (Luye Pharma Group, China), EndoTAG®-1 (Medigene) and LEP-ETU (NeoPharm) is less than 4 mol%. The lipids assembled into liposomes are generally inert in nature without therapeutic effects and the application of extra excipient materials in
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liposomes may raise cost burden and risk the safety concerns [17-19]. Thus, a stable liposomal formulation with high drug loading and biological activity is essential for combinatorial drug delivery. To address these limitations, one fascinating approach is the construction of dual functional nanocarriers that enable themselves self-assembly with therapeutic effects and further can synergistically enhance the antitumor activity by codelivery of incorporated drugs [20-23]. A representative example of dual functional noncarrier is d-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) by esterification of hydrophobic Vitamin E succinate and hydrophilic PEG1000 segment. The amphiphilic TPGS forms core-shell spherical nanostructure in aqueous medium, which can synchronously deliver hydrophobic agents with its own Vitamin E to tumor sites. Previous studies have established the synergistic efficacy of TPGS-based delivery system in a couple of in vitro and in vivo evaluations [24-27]. Moreover, drug-phospholipid conjugate based nanocarrier systems have been explored for the delivery of various chemotherapeutics [28,29]. Pedersen et al. [30] developed a novel active nanocarrier by conjugating chlorambucil drug with phosphatidylcholine, which assembled into unilamellar vesicles and displayed considerable cytotoxic potential against cancer cell lines. Prostaglandin-phospholipid conjugate was further synthesized and assembled forming vesicular structures in aqueous buffer with enhance cytotoxicities against HT-29 and Colo205 cells. Similarly, retinoid phospholipid conjugate-based liposomes were developed in 2010, which released retinoids under hydrolysis and displayed inhibition against colon cancer cells [31,32]. Recently, our group demonstrated the concept of dual drug-tailed phospholipid, replacing the two tails of the fatty acid chains by hydrophobic drug molecules and assembling itself into liposomes. For example, a liposomal formulation derived from dual chlorambucil-tailed phospholipid (DCTP) was developed to delivery chlorambucil, which exhibited significant antitumor efficacy with reduced side effects, highlighting their potential applications in combinatorial therapy [33]. Artesunate (ART), a naturally anti-malarial compound, shows anticancer activities against a variety of breast cancer, ovarian cancer and melanoma cancer cells [34]. Notably, it possesses the favorable selective property that is toxic to cancer cells but well safe towards normal cells, especially in case of combinational chemotherapy, which would benefit the biocompatibility after in vivo administration [35]. Based on this rational, we designed a novel active dual functional liposomal formulationbased on di-ART-PC conjugate with the aim to realize the precise codelivery of other payloads and synergistic prevention of tumor growth. PTX, as a first-line clinical anticancer drug, was passively loaded into the nanocarrier to formulate PTX/di-ART-PC liposomes with defined ratios by a typical dried thin-film technique. The physicochemical characterizations, stability and in vitro release behavior of
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2. Materials and Methods 2.1. Regents Artesunate (ART, purity ≥ 98%) was provided by Yuanye Bio-Technology Co., Ltd (Shanghai, China). Glycerylphosphorylcholine (GPC, purity ≥ 98%) was purchased from Innochem Technology Co., Ltd (Beijing, China). 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), N,N’-carbonyldiimidazole (CDI), tert-butyl diphenyl chlorosilane (TBDPSCl, purity ≥ 97%), tetra-butylammonium fluoride (TBAF) and dimethyl sulphoxide (DMSO) were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). ε-caprolactone, dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) and dichloromethane (DCM) were obtained from Juyou Scientific Equipment Co., Ltd (Nanjing, China). N,N’-dimethylformamide (DMF) and tetrahydrofuran (THF) were received from Sinopharm Chemical Reagent Co., Ltd (Nanjing, China). Unless otherwise stated, all the chemicals and reagents were of analytical grade. MCF-7 cells (human breast carcinoma), A549 cells (human lung carcinoma) and HepG-2 cells (human liver carcinoma) were gifted from the Chinese Academy of Sciences (Shanghai China). Secreted phospholipases A2 (sPLA2) was purchased from Mall Bio-Technology Co., Ltd (Nanjing, China). Fetal bovine serum (FBS), methyl tetrazolium (MTT), RPMI 1640 medium, cyanine (Cy5.5) and trypsin without EDTA were purchased from KeyGen Biotech Co., Ltd (Nanjing, China).
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2.2. Instruments Analytical high-performance liquid chromatography (HPLC, Agilent, CA) equipped with Agilent 1100 Infinity Quaternary HPLC System including a ZORBAX SB-C18 analytical column (4.6 × 150 mm) and a VWD UV-Vis detector. Mass spectrometry was carried out at Agilent 6540 instrument (MS, Agilent, CA). 1H NMR and 13C NMR spectra were performed on a Bruker DPX spectrometer at 500 MHz (Bruker, WI). 2.3. Synthesis of di-ART-PC conjugate Di-ART-PC prodrug was synthesized by covalent conjugation of artesunate (ART) to glycerophosphocholine (GPC) as shown in Scheme 1. The detailed steps are described below: Compound A: In the first step, ε-caprolactone (10 g, 0.087 mol) was added gradually
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to a solution of sodium hydroxide (NaOH, 3.5 g, 0.087 mol) in 100 mL water. After stirred at 25 °C for 12 h, the solution was extracted with ethyl acetate (EtOAc, 3 × 80 mL) and dried the solvent to get a white solid A (12.9 g, yield 98%). The chemical structure of compound A was checked via MS and 1H NMR as presented in supporting information (Figure S1 and S2). MS (m/z): calcd for C6H12O3, 132.08; found, 155.0 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 5.33 (s, 1-H, H/1), 3.67 (t, 2-H, H/2, J = 6.5 Hz), 2.37 (t, 2-H, H/6, J = 7.4 Hz), 1.68 (m, 2-H, H/3), 1.61 (m, 2-H, H/5), 1.43 (m, 2-H, H/4) ppm.
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Compound B: In the second step, to a solution of compound A (2 g, 15 mmol) and imidazole (1.02 g, 15 mmol) in anhydrous DMF (30 mL), TBDPSCl (4.12 g, 15 mmol) was added gradually and the reaction was stirred at 25 °C overnight. After diluting the resultant mixture with DCM (80 mL) and subsequently washing with deionized water (30 mL × 3), the solvents were first evaporated and dried by anhydrous Na2SO4, followed by purification via silica column chromatography using (EtOAc: hexane, 1:3) as an eluent provided compound B as a colorless viscous liquid (4.32 g, yield: 77%). The chemical structure of this step was confirmed by MS and 1H NMR as presented in supporting information (Figure S3 and S4). MS (m/z): calcd for C22H30O3Si, 370.20; found, 371.20 [M+H]+, 393.18 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 7.76 (d, 4-H, H/1,1’,5,5’, J = 6.5 Hz), 7.44 (t, 6-H, H/2,2’,3,3’,4,4’, J = 7.4 Hz), 3.75 (t, 2-H, H/9, J = 6.5 Hz), 2.41 (t, 2-H, H/13, J = 7.5 Hz), 1.71-1.65 (m, 2-H, H/10), 1.54-1.48 (m, 2-H, H/12), 1.34-1.29 (m, 2-H, H/11), 1.14 (s, 9-H, H/6,7,8) ppm.
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Compound C: In the third step, 0.87 g/5.4 mmol of CDI was mixed with a solution of compound B (2 g, 5.4 mmol) dissolved in DCM (15.00 mL) and stirred at room temperature for 4 h. Meanwhile, 0.69 g/2.7 mmol of GPC dissolved in anhydrous DMSO (20.00 mL) was stirred with DBU (0.39 g, 2.6 mmol) for 4 h at 35 °C. Then, the two solutions were combinedand nightlystirred at room temperature. The reaction progress was observed through TLC (20% methanol in CH2Cl2 as an eluent, UV light at 254 nm). Purification of the product was carried out by silica column chromatography using CH2Cl2/CH3OH (solvent A: CH2Cl2:CH3OH, 10:1; solvent B: CH2Cl2:CH3OH:H2O 65:25:4) as an eluent, provided C as a white solid (3.5 g, yield: 33%). The structure of compound C was confirmed by MS and 1H NMR as presented in supporting information (Figure S5 and S6). MS (m/z): calcd for C52H76NO10PSi2, 961.47; found, 962.47 [M+H]+, 984.45 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 7.69 (d, 8-H, H/3,3’,4,4’,6,6’,10,10’, J = 6.5 Hz), 7.41 (t, 12-H, H/1,1’,2,2’,5,5’, 7,7’,8,8’, 9,9’, J = 7.0 Hz), 5.24 (d, 2-H, H/20, J = 5.5 Hz), 4.44 (t, 2-H, H/22, J = 7.4 Hz), 4.16 (m, 1-H, H/19), 3.99 (t, 2-H, H/23, J = 7.5 Hz), 3.93 (t, 4-H, H/14,14’, J = 7.0 Hz), 3.72 (d, 1-H, H/21, J = 7.4 Hz), 3.29 (s, 9-H, H/24, 25,26), 2.33-2.26 (m, 4-H,
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Compound D: In the final step, the protection of hydroxyl (-OH) group in compound C by TBDPSCl agent was deprotected using TBAF solution. Briefly, TBAF (1 M, 2 mL) of was added to compound C solution (1 g, 1.0 mmol) in THF (30 mL). The reaction mixture was continuously stirred for 2 h at ambient temperature, while their completion was observed with TLC (solvent: 20% methanol in CH2Cl2, UV light at 254 nm). To this obtained product without further purification, ART (0.79 g, 2.78 mmol), EDC.HCl (0.53 g, 2.80 mmol) and DMAP (0.15 g, 1.22 mmol) were added in 30 mLof DMSO and over nightly stirred at 25 °C. The mixture was subjected to chromatographic purification, which first eluted with solvent A (CH2Cl2:CH3OH, 10:1) and then with solvent B (CH2Cl2:CH3OH:H2O, 65:25:4), afforded the white solid compound D (di-ART-PC, 0.31 g, yield: 25%). The structure of di-ART-PC was verified by MS, 1H NMR and 13C NMR. (0.31 g, yield: 25%). MS (m/z): calcd for C57H92NO24P, 1217.5; found, 1218.7 [M+H]+, 1240.7 [M+Na]+. 1H NMR (500 MHz, CDCl3): δ 5.79 (d, 2-H, H/10,10’, J = 9.8 Hz), 5.45 (s, 2-H, H/8,8’), 5.22 (d, 2-H, H/26, J = 5.5 Hz), 4.33 (t, 2-H, H/29, J = 10.5 Hz), 4.15 (m, 1-H, H/27), 4.09 (t, 4-H, H/20,20’, J = 6.0 Hz), 3.97 (t, 2-H, H/30, J = 6.5 Hz), 3.79 (d, 2-H, H/28, J = 7.4 Hz), 3.36 (s, 9-H, H/31,32,33), 2.78-2.50 (m, 10-H, H/13,13’,21,21’,22,22’), 2.46-2.26 (m, 8-H, H/18,18’,24,24’), 2.10-1.58 (m, 24-H, H/4,4’,5,5’,14,14’,15,15’,17,17’,23,23’), 1.44 (s, 6-H, H/7,7’), 1.40-1.02 (m, 6-H, H/2,2’,3,3’,11,11’), 0.98 (d, 6-H, H/1,1’, J = 5.7 Hz), 0.87 (d, 6-H, H/12,12’, J = 6.9 Hz) ppm. 13C NMR (500 MHz, CDCl3): δ 172.37, 171.62, 170.65, 103.91, 91.65, 90.98, 79.60, 70.21, 66.04, 64.08, 62.99, 62.45, 58.71, 54.02, 51.06, 44.72, 36.71, 35.73, 33.63, 33.33, 31.28, 28.69, 28.29, 27.66, 25.46, 24.88, 24.77, 24.04, 23.89, 21.45, 19.69, 11.52 ppm.
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2.4. Critical micelle concentration (CMC) of di-ART-PC conjugate Pyrene fluorescent probe-technique was used to determine the critical micelle concentration (CMC) of di-ART-PC conjugate on a HJYFL3-211-TCSPC spectrometer (HORIBAJobin Yvon, France). In detail, di-ART-PC conjugate was solubilized in distilled water and diluted a series of concentrations from 70 µg/mL to 5µg/mL, where, each tube contained a total of 1 mL solution. A pyrene solution having a concentration of 5×10-5 M in acetone was added into di-ART-PC solutions in different tubes. After equilibration at 25 °C for 24 h, the samples were subjected to HJYFL3-211-TCSPC spectrometer to record fluorescence spectra of each solution. The excitation wavelength of the fluorescence scan was 334 nmwith the excitation slit of 5.0 nm and the emission slit was set to 2.5 nm. The CMC value of di-ART-PC conjugate was determined from the intensity ratio of the first vibronic band (I1, λ1 is
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2.5. Characterization of di-ART-PC and PTX/di-ART-PC liposomes Both di-ART-PC and PTX/di-ART-PC liposomes were formulated by employing reverse-phase evaporation technique according to the previous studies [36-38]. For di-ART-PC liposomes, di-ART-PC conjugate (10 mg, 8.21 mmol) was solubilized in 30 mL dichloromethane, following the addition of PBS buffer (pH 7.4, 10 mL) and the resulted emulsion was ultrasonicated by Ymnl-150Y for 30 min. DCM was removed by evaporation, and the liposomes were rotated at 50 °C for 1 h in rotary evaporator. Finally, the di-ART-PC liposomes were homogenized by a 0.22 um filter membrane with a micro-extruder at 50 °C. For PTX/di-ART-PC liposomes, di-ART-PC and PTX in different ratios (Table 1) were dissolved in DCM (30 mL), and then added 10 mL of PBS buffer (pH 7.4). Afterward, Ymnl-150Y supersonic probe was applied to sonicate the obtained emulsion for 30 min, followed by evaporation at rotary evaporator. The residual solvent, un-loaded PTX and un-assembled di-ART-PC were removed by dialysis in PBS (pH 7.4, 200 mL) for 12 h to obtain a final suspension of the PTX/di-ART-PC liposomes. Additionally, the conventional ART and PTX-loaded liposomes composed of lecithin/Chol/DSPC-PEG (90:10:5 Mol% ratio) (combo-liposomes) were prepared as similar as the above procedure to compare their in vitro antitumor effects with PTX/di-ART-PC liposomes. Size and zeta-potential of the afforded liposomes were measured by DLS. The morphology of di-ART-PC liposomes was analyzed by using transmission electron microscopy (TEM). Samples were prepared by dropping 10 µL of liposomes solution on a 200-mesh carbon film copper grid, air-drying and negatively staining (phosphomolybdic acid, 2% w/v). Observation was carried out by JEM-2100 TEM (JEOL, Japan) at 200 kV. 2.6. In vitro release of ART and PTX The drug release profiles of PTX/di-ART-PC liposomes were estimated in simulated neutral (PBS, pH 7.4) and weakly acidic (PBS, pH 5.0) release mediums. For release experiment, PTX/di-ART-PC liposomal suspension (3 mL) was incubated in a dialysis bag (MWCO, 3500) in a tube containing release medium (30 mL) supplemented with 1% Tween 80 at 37 °C. The tubes were kept in Shaker Incubator LSI-100B under shaking condition (100 rpm). At predetermined time points, the release medium (1 mL) containing released drugs was taken and immediately substituted with an equivalent volume of fresh media. PTX and ART contents were analyzed by means of HPLC under eluting solvents of 80% MeCN in water (0.1%
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TFA). Furthermore, the release of di-ART-PC liposomes was performed with secretory phospholipase A2 (sPLA2) according to the reported literature [39,40]. The catalytic release was initiated by adding 2 µL of a 45 µM sPLA2 stock solution to 20 mL of 25 µM di-ART-PC liposomes at 37 °C equilibrated 20 min prior to addition of the enzyme. The release of ART was measured by HPLC with 20 µL of solution sample taken every ten minutes. All experiments were repeated thrice and the results were expressed as the mean ± standard deviation.
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2.7. In vitro cellular uptake The cellular uptake property of PTX/di-ART-PC liposomes labeled by Cy5.5 dye was analyzed via confocal laser scanning microscopy (CLSM) after incubation with MCF-7 cells. The Cy5.5-labelled di-ART-PC liposomes were formulated by reverse-phase evaporation method as described above under light protection conditions. MCF-7 cells were treated with Cy5.5-labelled di-ART-PC liposomes (5 µg Cy5.5/mL) for two different time periods (2 h and 4 h) at 4 °C or 37 °C, respectively. After washing the treated cells twicewith PBS and fixing by 4% paraformaldehyde, the cells were stained for 15 min with 4,6-diamidino-2-phenylindole (DAPI) at room
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2.8. In vitro cytotoxic activity The cell viability of PTX/di-ART-PC liposomes versus MCF-7, A549 and HepG-2 three different cancer cell lines was measured by MTT assay using free ART, free PTX, and conventional ART/PTX-loaded lecithin liposomes (combo-liposomes), as controls. In the experimental protocol, 100 µL of cell suspensions with the density of 7.5×103 cells per well was seeded in plates (96-well) and cultured at 37 °C overnight. Then, cells in each well were treated with various concentrations of PTX/di-ART-PC liposomes and control solutions, and incubated for additional 24 h under standard incubatory condition. In order to evaluate the cell viability after drug treatment, MTT solution (20 µL, 1 mg/mL,) was supplemented to all wells and continued incubation for 4 h. After the removal of medium, dimethyl sulfoxide (150 µL) was added to solubilize the developed crystals in the wells for 5 min. Finally, the microplate reader (Multiskan EX, Thermo Scientific, Waltham, MA) was applied to check the absorbance at 570 nm and to determine the cell viability as follows.
Cell viability (%)=
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ODtreated represents the optical density (OD) from treatment groups and ODcontrol
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indicates the OD from growth medium, whereas, ODblank is the OD from MTT solution lacking cells, respectively. Combination index (CI) is commonly used to analyze the synergistic, antagonistic or additive effects of two drugs [41,42]. The ART and PTX drug combinatorial from PTX/di-ART-PC liposomes and combo-liposomes were used to calculate the CI values, where CI > 1 indicates antagonism; CI = 1 represents additive effect and CI < 1 designates synergistic effects. CI was calculated by the following formula: C1 C2 CI = + Cx1 Cx2 Whereas, C1 and C2 is the concentration of drug 1 and drug 2 to produce a certain effect in combination, while Cx1 and Cx2 represents the concentration of drug 1 and 2 generating identical effect alone [43-45]. GraphPad sigmoid curves (efficacy–dose–response relationships) were used to determine the degree of effect foreach drug for calculating CI [46-49]. 2.9. Cell apoptosis assay MCF-7 cells at a concentration of 1×105 cells per well were cultured in 12-well plates. Post-incubation for 24 h, the cells were treated with free ART, free PTX, ART/PTX mixture, combo-liposomes and PTX/di-ART-PC liposomes, respectively at
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an equal drug concentration of 5 µg/mL. Then, the cells were incubated for 24 h at 37 °C. After incubatory period, the cells were washed twice with PBS, followed by treatment with binding buffer and staining through Annexin-V and PI (propidiumiodide) under dark (10 min). Finally, the cells were examined for apoptosis
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2.10. Hemolytic toxicity The hemolytic toxicity of di-ART-PC liposomes (both blank and PTX-loaded) was conducted by incubating with human red blood cells (RBCs) in order to check their blood compatibility. Briefly, 10 mL of fresh whole blood from healthy volunteers was centrifuged on 1500 rpm at 4 °C for 10 min to take away the supernatant. The precipitated red blood cells were washed thrice with saline till the supernatant was clear. The obtained red blood cells were mixed with normal saline to prepare a 2% erythrocyte suspension (2 mL of RBCs and normal saline to 100 mL). Then, 25 µL aliquots of erythrocyte suspension were exposed to blank di-ART-PC liposomes at concentrations of 5, 25 and 50 µg/mL, and to PTX/di-ART-PC liposomes at PTX concentrations of 1, 5 and 10 µg/mL. The samples were incubated in a shaker for 4 h at 37 °C and then centrifuged at 1500 rpm for 15 minutes. Supernatants were collected and the absorbance of hemoglobin was measured at 545 nm through UV-Vis
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spectrometer. The selected normal saline and deionized water causing zero hundred percent hemolysis were used as negative and positive controls, respectively. The hemolysis ratio lower than 5% was interpreted safe. The % hemolysis rate was determined by the following formula: Aexp − Aneg Hemolysis rate % = × 100 Apos − Aneg where, Aexp is the absorbance of experimental group, where, Apos and Aneg are absorbance of positive and negative controls, respectively.
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3. Results and Discussion 3.1. Synthesis of di-ART-PC conjugate The di-ART-PC conjugate was synthesized by the covalent conjugation of ART with PC under CDI/DBU catalytic system as elucidated in Scheme 1. The high purity (96%) di-ART-PC prodrug with an overall yeild of 25% was obtained as indicated in Figure 1A (inset). The MS and 1H NMR spectra of the intermediates (A-C) are presented in supporting information (Figure S1-S6). The MS data of di-ART-PC conjugate shows the molecular iron peaks at 1218.7 (m/z, [M+H]+) and 1240.7 (m/z, [M+Na]+), that are in agreement with the calculated value (C57H92NO24P 1217.7, Figure 1A). 1H NMR spectrum (Figure 1B) of the di-ART-PC conjugate displayed signals from methyl protons of artesunate at 1.44 ppm, 0.98 ppm and 0.87 ppm, along
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1C). All these experimental results clearly established the successful synthesis of di-ART-PC prodrug.
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Scheme 1. Synthetic procedure of di-ART-PC conjugate. Reagents and conditions: (a) NaOH/H2O, RT, 12 h, 98%; (b) TBDPSCl, imidazole, DMF, RT, 12 h, 77%; (c) L -α-glycerophosphocholine, CDI, DBU, CH2Cl2, DMSO, RT, 12 h, 33%; (d) TBAF, THF, RT, 4 h, 96%; (e) ART, EDC.HCl, DMAP, THF/DMSO, RT, 12 h, 25%.
Scheme 2. Illustration of the self-assembly of di-ART-PC into liposomes for PTX loading.
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ACCEPTED MANUSCRIPT Figure 1. Characterization of di-LA-PC conjugate: (A) Mass spectrum, (inset) HPLC profile of di-ART-PC eluted with 80% MeCN in water (0.1% TFA in 18 min at 10.237 min; (B) 1H NMR (500 MHz, CDCl3) and (C) 13C NMR (500 MHz, CDCl3) spectra.
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3.2. Critical micelle concentration (CMC) of di-ART-PC conjugate Self-assembly ability of the di-ART-PC conjugate was verified by its critical micelle concentration (CMC), determined by pyrene fluorescent probe method. The existence of hydrophobic ART and hydrophilic GPC moieties facilitated the rapid assembly of di-ART-PC conjugate in aqueous medium. The intensity ratio (I3/I1) was recorded as a function of di-ART-PC concentration (Figure 2). Due to high sensitivity of pyrene to the surrounding polar medium, it readily showed a sharp shift in I3/I1 (emission intensity ratio) and hence the CMC value could be determined. Under lower di-ART-PC concentration, no substantial change in I3/I1 value was observed but upon the enhancement of conjugate concentration, the ratio of I3/I1 rapidly augmented
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which could be attributed to the deposition of pyrene in hydrophobic atmosphere. Based on the sharp change in the curve, CMC value was calculated as 22 µg/mL,
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highlighting the successful self-assembly of di-ART-PC conjugate in aqueous media.
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Figure 2. Relationship between the fluorescent intensity ratio (I3/I1) and di-ART-PC concentration dispersed in water. The CMC value is about 22 µg/mL. 3.3. Characterization of di-ART-PC liposomes The developed amphiphilic di-ART-PC conjugate having two ART moieties conjugated with phospholipid demonstrated proficient self-assembly ability in aqueous environment, resulted into di-ART-PC liposomes. The size and morphology of the liposomes were investigated by DLS and TEM. As illustrated in Supporting information (Figure S7), the blank di-ART-PC liposomes have small size (163.05 nm) and a narrow distribution (PDI: 0.283) along with negatively charged surface (zeta potential of -23.37 mV) in water, indicating their promising stability. In addition,
ACCEPTED MANUSCRIPT TEM images revealed the spherical nanostructure of the di-ART-PC conjugate assembled liposomes with a diameter of about 100 nm, which is close to that determined by DLS. Di-ART-PC conjugate and PTX at different ratios were used to attain optimized
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liposomal formulation (Table 1). As shown in Table 1, the di-ART-PC conjugate to PTX in the ratio of 10:2 (mole ratio) showed higher DLE (83.9%) and a favorable DLC (10.52%) compared to the already reported liposomes [50,51]. In addition, the liposomes loaded with different concentrations of PTX were incubated in PBS (pH 7.4) solution for 1-10 days under dialysis bag (MWCO, 3500), while their stability and drug encapsulation rate were determined as a function of time. As shown in Figure S8, the liposomes developed form the initial feed molar ratios of conjugate to PTX (10:0.5, 10:1, 10:2) showed better stability, where the drug loaded rate changes were less than 10%. Due to the optimal particle size, stability, and drug loading efficiency, the drug to
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carrier ratio of 2:10 was selected to prepare optimized PTX/di-ART-PC liposomes and used in the following experiments. The average size of the optimized PTX/di-ART-PC liposomes slightly increased to 174.60 nm (Figure 3), versus blank di-ART-PC liposomes as described above. This may be due to the fact that the drug PTX is loaded into the liposome and the inert space of these liposomes is increased. Besides, the storage stability of PTX/di-ART-PC liposomes under simulated physiological
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incubation. Overall, the di-ART-PC conjugate has efficient self-assembly ability benefiting from their amphiphilicity, as well as demonstrated promising loading efficiency of other anticancer agents.
Figure 3. Characterization of PTX-loaded di-ART-PC liposomes: (A) size and zeta-potential measured by DLS, and (B) TEM image.
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di-ART-PC:PTX
b
PDI
Zeta-potential
c
DLC %
d
DLE %
10:0.5
167.05±3.58
0.261
-21.55
2.83
83.1
10:1
169.25±3.64
0.256
-18.11
5.43
82.0
10:2
174.60±4.21
0.224
-17.30
10.52
83.9
10:5
180.35±5.03
0.228
-15.84
19.31
68.3
b
Data are given mean ± standard deviation (n=3); PDI: Polydispersity index; cDLC:
Drugloading capacity; dDLE: Drug loading efficiency.
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Size
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3.4. In vitro release study In vitro release of ART and PTX from PTX/di-ART-PC liposomes was investigated by dialysis (MWCO 3500) under two different simulated pH conditions (PBS pH 7.4 or pH 5.0). As shown in Figure 5, no significant ART and PTX release
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(less than 20%) was observed after a 72 hour incubation in PBS pH 7.4, which demonstrates the stable liposomal nanostructure of di-ART-PC liposomes as well as stable encapsulation of PTX within the liposomes. This behavior of the liposomes is desirable for the good stability under biological environment. Under weakly acidic environment (PBS, pH 5.0), rapid release rate of both PTX and ART were found during the first 24 h and the overall sustained release of 74% and 63.5%, respectively
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were reached over the next 48 h. It is supposed that the PTX/di-ART-PC liposomes dissociate and subject to acidic hydrolysis to sustainably release parent drug ART. As shown in Figure S10B, significant ART peak at 2.783 min was observed along with a peak at 7.431 min of ART-lysophospholipid. After 2 h incubation,
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appropriate 80% of ART was released from di-ART-PC liposomes (Figure S10C). The results clearly demonstrated that di-ART-PC liposomes were effectively degraded to
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release free ART in the presence of catalytic sPLA2. Conclusively, these PTX-loaded di-ART-PC liposomes is relatively stable without premature drug release under neutral condition, but could rapidly release antitumor agents in an acidic or lipases environment, leading to killing of the tumor cells effectively.
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Figure 5. In vitro release of ART and PTX from PTX/di-ART-PC liposomes under different simulated pH conditions for 72 h.
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3.5. Cellular uptake of di-ART-PC liposomes The cellular uptake behavior of di-ART-PC liposomes in MCF-7 cells was investigated by confocal laser scanning microscopy. For ease of observation, near-infrared fluorescence (NIRF) Cy5.5 was used to label di-ART-PC liposomes, while the cellular nuclei were stained by DAPI with blue fluorescence. As shown in Figure 6, the internalization of di-ART-PC liposomes into the MCF-7 cells can be clearly observed. The nucleus appears blue, as opposed to the cytoplasmic region (red), demonstrating that the di-ART-PC liposomes can enter into cytoplasm. These observations indicate that the di-ART-PC liposomes (red stained) are predominantly distributed throughout the cytoplasm. It has been reported that the mechanism of particle uptake is phagocytosis, liquid phase pinocytosis or receptor-mediated endocytosis. The main internalization mechanism of 100 nm particles is endocytosis, supporting the distribution of the liposomes into the cytoplasm as well as in the nucleus. Cy5.5-labelled di-ART-PC liposomes showed more definitive uptake during the incubation for 2 hours at 37 °C compared to 4 °C, and cellular uptake increased within 4 hours. It is therefore suggested that di-ART-PC liposomes encapsulated with PTX could enter into the cells and could effectively accumulate in the nucleus to improve the therapeutic effects in cancer therapy.
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Figure 6. Confocal laser scanning microscope analysis of Cy5.5-labelled di-ART-PC liposomes, showing their uptake into MCF-7 cells at 37 °C or 4 °C for 2 h and 4 h. Cell nuclei were stained by DAPI (blue). Scale bar: 25 µm.
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3.6. In vitro cytotoxicity of PTX/di-ART-PC liposomes Cytotoxicity is a major concern when developing delivery systems for anticancer drugs. Therefore, the in vitro cytotoxicity of PTX/di-ART-PC liposomes was determined by standard MTT assay and compared with different free drug and drug combinations against MCF-7, HepG-2 and A549 cells. The cytotoxic activity of PTX/di-ART-PC liposomes enhanced with the increase in drug concentration. Importantly, the PTX/di-ART-PC liposomes demonstrated significant anti-tumor effects that are higher than free ART, free PTX and conventional ART/PTX-loaded lecithin liposomes (combo-liposomes) (Figure 7). These findings suggested that PTX/di-ART-PC liposomes have better anticancer effects. As indicated in Table 2, the half maximal inhibitory concentration (IC50) values of PTX/di-ART-PC liposome versus MCF-7, HepG-2and A549 cancer cells were determined to be 2.9 ± 0.11 µg/mL, 4.3 ± 0.22 µg/mL, 3.0 ±0.14 µg/mL, respectively. These results were far significantly lower than control groups (Table 2). In this study, the combination index CI values of the PTX/di-ART-PC liposomes for MCF-7, HepG-2 and A549 cells were 0.753 µg/mL, 0.724 µg/mL and 0.606 µg/mL,
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combinatorial antitumor drug delivery. Based on the above data, PTX/di-ART-PC liposomes effectively delivered both antitumor agents to cancer carcinomas, and produced improvement in in vitro cytotoxic effects synergistically.
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Figure 7. Cell viability of free ART, free PTX, conventional combo-liposomes, and PTX/di-ART-PC liposomes. Error bar represent mean ± standard deviation (n = 4).
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Table 2. IC50 Values of ART, PTX, combo-liposomes, and PTX/di-ART-PC liposomes against MCF-7, Hepg2 and A549 cancer cells after 24 h incubation. Error bar represent mean ± standard deviation (n = 3). IC50 (µg/mL)
Formulation
MCF-7
HepG-2
A549
ART
34.6± 0.13
57.2± 0.19
37.5± 0.15
9.3± 0.21 4.5± 0.41
13.4± 0.31 6.1± 0.26
11.8± 0.27 4.1± 0.33
2.9± 0.11
4.3± 0.22
3.0± 0.14
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PTX Combo-liposomes PTX/di-ART-PC
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Table 3. Combination index values of combo-liposomes and PTX/di-ART-PC liposomes in three cancer cell lines. CI (µg/mL) Cells MCF-7 HepG-2 A549
Drug combination Combo-liposomes
PTX/di-ART-PC
0.915
0.753
0.924 0.909
0.724 0.606
3.7. Cell apoptosis assay Apoptotic assay was applied to evaluate the killing mechanism of MCF-7 cells treated with free ART, free PTX, conventional combo-liposomes and PTX/di-ART-PC
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control. The apoptotic results were shown in Figure 8. Among the treatment groups, PTX/di-ART-PC liposomes exhibited higher percentage of cells in early and late apoptosis compared to control groups at the same equivalent concentration. This apoptotic result is consistent with the MTT data. More specifically, the percentages of early apoptotic cells referred as Q4 (annexin positive/PI negative) for ART, PTX, conventional combo-liposomes and PTX/di-ART-PC liposomes were 2.38 ± 2.19%, 2.70 ± 2.56%, 4.33 ± 3.01% and 4.02 ± 2.89%, whereas those of late apoptotic cells (referred as Q2, annexinand PI double positive) were 6.61 ± 3.32%, 9.77 ± 2.19%, 14.0 ± 2.41% and 18.1 ± 2.57%, respectively (Figure 9). These results clearly demonstrated that PTX/di-ART-PC liposomes could increase the PTX-induced
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apoptosis upon their entrapment into active di-ART-PC nanocarrier compared to the plain PTX at equivalent concentration.
Figure 8. Flow cytometry induced apoptotic analysis of MCF-7 cells by ART, PTX, conventional combo-liposomes and PTX/di-ART-PC liposomes, at the same concentration (5 µg/mL) for 24 h. Nontreated cells were exploited as control. In each flow cytometry plot, Q1 refers to necrosis, Q2 late apoptosis, Q3 early apoptosis and Q4 viable cell.
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Figure 9. Quantitative analysis of MCF-7 cells apoptosis. Data are expressed as mean ± SD. 3.8. Hemolytic toxicity An important criterion for in vivo application is the blood compatibility of a nanocarrier after administration that is assessed by hemolysis study. Thus, the
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hemocompatibility of unloaded di-ART-PC and PTX/di-ART-PC was evaluated at different concentrations by incubating with human normal red blood cells (RBCs) as an in vitro toxicity model. The changes in hemolysis rate as a function of concentration induced by the treatment groups were presented in Figure 10. After incubation for 4 hours at 37 °C, the unloaded and PTX-loaded di-ART-PC liposomes were found to be non-hemolytic (<5%). The data significantly indicated that the di-ART-PC and PTX/di-ART-PC nanocarrier systems have good biocompatibility for in vivo administration.
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Figure 10. Hemolysis of unloaded di-ART-PC and PTX/di-ART-PC after 4 h incubation with blood erythrocytes at 37 °C. Each value represents the mean ± SD of three experiments.
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4. Conclusion In summary, a successful formulation based on active di-ART-PC liposomal nanocarrier platform was developed for PTX delivery for cancer treatment. Hydrophobic PTX was effectively encapsulated into the core of liposome with an impressive drug loading capacity and significant stability. The lower and uniform distributed size (<200 nm) of the PTX/di-ART-PC liposome demonstrated efficient internalization into tumor cells. In vitro cytotoxicity assay and apoptosis assays confirmed the effectiveness of the liposomes that has higher tumor inhibition and a good synergistic therapeutic effect (CI value < 1). All these results suggested that PTX/di-ART-PC liposomes may be a promising combination of anti-cancer regimens that could effectively improve the therapeutic effects. Moreover, the active dual functional di-ART-PC assembled liposomes can be a great promising candidate to synergistically delivery of anticancer therapeutics, thus offering a versatile platform in combinatorial nanomedicines. Acknowledgement The current work was funded by the Major National Science and Technology Program of China for Innovative Drug (2017ZX09101002-001-004), the National Natural Science Foundation of China (No. 51373034), the Department of Science & Technology of Jiangsu Province, China (Projects BA2013037, BY2015070-11) and the Priority Academic Program Development of Jiangsu Higher Education
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Conflicts of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Paclitaxel encapsulated in artesunate-phospholipid liposomes for combinatorial delivery”.
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