- Email: [email protected]
Polymer–lipid hybrid nanoparticles-based paclitaxel and etoposide combinations for the synergistic anticancer efficacy in osteosarcoma
Accepted Manuscript Title: Polymer-lipid hybrid nanoparticles-based paclitaxel and etoposide combinations for the synergistic anticancer efficacy in osteosarcoma Author: Rui Duan Caiyan Li Fan Wang Jin-Chu Yangi PII: DOI: Reference:
S0927-7765(17)30557-X http://dx.doi.org/doi:10.1016/j.colsurfb.2017.08.042 COLSUB 8798
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-12-2016 21-8-2017 23-8-2017
Please cite this article as: R. Duan, C. Li, F. Wang, J.-C. Yangi, Polymer-lipid hybrid nanoparticles-based paclitaxel and etoposide combinations for the synergistic anticancer efficacy in osteosarcoma, Colloids and Surfaces B: Biointerfaces (2017), http://dx.doi.org/10.1016/j.colsurfb.2017.08.042 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.
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1. Paclitaxel and etoposide-loaded lipid-polymer hybrid nanoparticles was prepared for
enhanced efficacy in osteosarcoma 2. PE-LPN exhibited a remarkably higher apoptosis cell death of cancer cells
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3. PE-LPN showed a remarkable tumor regression effect and exhibited a 2-fold superior
efficacy than free drugs
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4. Combination of drugs in a LPN carrier could greatly improve the therapeutic property of
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Polymer-lipid hybrid nanoparticles-based paclitaxel and etoposide combinations for the synergistic anticancer efficacy in osteosarcoma
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Rui Duan1#, Caiyan Li2#, Fan Wang3*, Jin-Chu Yangi4*,
Department of oncology, The first People’s Hospital of Jingmen, Jingmen, Hubei 448000, China
2
Department of Clinical laboratory, The second People’s Hospital of Jingmen, Jingmen, Hubei
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1
3
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448000, China
Department of orthopaedics, The first People’s Hospital of Jingmen, Jingmen, Hubei 448000,
China
Department of hand surgery, Luoyang Orthopedic Hospital of Henan Province, Henan, 471002,
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4
China
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#These two authors contribute to this work equally
Corresponding author:
Dr. Jin-Chu Yangi, Department of hand surgery, Luoyang Orthopedic Hospital of Henan Province, No.233, Qiming South Road, Luoyang, 471002, Henan, P.R.China Tel&Fax: 0086-379-63546996 Email: [email protected]
Dr. Fan Wang, Department of orthopaedics, Jingmen No.1 People’s Hospital, No.67 of Xiangshan Road, Jingmen, Hubei 448000, China Tel/Fax: 0086- 07242305966 [email protected] 1 Page 3 of 32
Abstract In this study, paclitaxel and etoposide-loaded lipid-polymer hybrid nanoparticles (PE-LPN) was
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successful prepared and evaluated for physicochemical and anticancer effect. Nanosized PE-LPN was obtained with a perfect spherical morphology. PE-LPN exhibited a controlled release of two
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drugs in a sequential manner. The nanoparticles exhibited a typical endocytosis-mediated cellular uptake in cancer cells. The ratiometric combination of paclitaxel (PTX) and Etoposide (ETP)
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were significantly more cytotoxic than individual drugs. Importantly, superior cytotoxic effect
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was observed for dual-drug-loaded PE-LPN than cocktail combination at a much lower dose. Similarly, PE-LPN exhibited a significantly higher apoptosis of cancer cells (~45%) compared to
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that of any other groups with higher caspase-3 & -8 activity. Importantly, PE-LPN showed a remarkable tumor regression effect and exhibited a 2-fold superior efficacy than free drugs. PE-
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LPN treated group showed significantly less Ki-67 positive cells (less than 25%) than PTX/ETP
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and single drug treated groups, suggesting less active cell proliferation and a considerably higher
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tumour growth inhibition effect. The results collectively showed that combination of drugs could greatly improve the therapeutic property of chemotherapeutic drugs. By combining ETP with PTX (a powerful anticancer drug) in a polymer–lipid hybrid nanoparticle system, therapeutic efficacy could be improved in osteosarcoma treatments.
Keywords Osteosarcoma, paclitaxel, etoposide, apoptosis, nanoparticles
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Introduction Osteosarcoma is one of the common forms of bone cancer affecting adult people aging between
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10 and 24 years [1,2]. Like the osteoblasts in normal bone, the cells that form this cancer make bone matrix. But the bone matrix of an osteosarcoma is not as strong as that of normal bones.
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Osteosarcoma is a bone cancer that typically develops in the shinbone (tibia) near the knee, the thighbone (femur) near the knee, or the upper arm bone (humerus) near the shoulder.
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Osteosarcoma tends to develop during growth spurts in early adolescence. This may be because
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the risk of tumors increases during this period of rapid bone growth. It accounts for nearly 60% of all bone cancers in Children and adults. It is diagnosed mostly in first 2 decades of their life
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term. It accounts for nearly 60% of all bone cancers in children and adults and is typically diagnosed in the first two decades of life. Despite significant advancements in the diagnosis and
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treatment of osteosarcoma, the 5-year survival rate is less than 65%, and metastatic cancer
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accounts for less than 20% of cases [3]. Surgery and conventional chemotherapy are the standard
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treatment for this disease, and the cure rate increases when chemotherapy is combined with surgery. Several classes of single anticancer drugs have been indicated for the treatment of osteosarcoma; however, failure of the chemotherapy regimen is a major factor in unsuccessful treatment [4,5]. Failure mainly results from drug resistance in patients with advanced-stage cancer. The complex microenvironment of cancer cells coupled with drug resistance contributes to the failure of chemotherapy based on a single drug [6,7]. Therefore, combination chemotherapy has been used to improve the therapeutic efficacy of drugs while simultaneously reducing drug-related side effects. Combination therapy is effective for treating drug-resistant cancers, improving therapeutic efficacy, and minimizing adverse effects [8]. Using more than two drugs that target different 3 Page 5 of 32
pharmacological pathways could alter genetic barriers, including cancer cell mutations and adaptations, and suppress drug efflux receptors while acting in a synergistic manner. Therefore, the use of a combination of anticancer drugs could result in greater therapeutic efficacy and
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target selectivity [9,10]. Here we investigated the suitability of a combination of paclitaxel (PTX) and etoposide (ETP) for treating osteosarcoma. PTX acts by stabilizing microtubules and
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blocking cancer cells at the G2/M phase, provoking cell apoptosis. In addition, PTX can enhance
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expression of nuclear factor-B (NF-B), a transcription factor that regulates various physiological processes, including inflammation, differentiation, and apoptosis [11,12]. ETP stabilizes covalent
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enzyme–DNA complexes that play important roles in the catalytic cycle of topoisomerase II. The accumulation of cleaved enzyme complex leads to a permanent break in the DNA strand,
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resulting in chromosomal translocation and eventual cell death [13,14].
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The anticancer efficacy of a combination of drugs could be severely hindered by the acute and
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chronic toxicity of the individual drugs. In addition, the therapeutic efficacy of two drugs could
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be hampered by poor pharmacokinetic performance, biodistribution, and membrane transport properties, which may lead to poor internalization into tumor tissues [15]. Therefore, it is important to improve the physicochemical properties and anticancer efficacy of dual drugs while limiting their side effects. In this regard, nanoparticles may be used to circumvent problems associated with drug delivery and improve pharmacokinetic parameters [16]. In particular, lipidpolymer hybrid nanoparticles (LPN) offer many attractive features for improving the anticancer efficacy of chemotherapeutic combinations. LPN offers high systemic stability, high drug loading, high protection of encapsulated compounds, and prolonged blood circulation. Nanosized particles will preferentially accumulate in tumor tissues via the enhanced permeation and retention (EPR) effect [17,18]. In addition, nanosized carriers consisting of naturally derived 4 Page 6 of 32
materials evade the reticuloendothelial system (RES) and inhibit systemic clearance, thereby prolonging drug circulation. Therefore, we used a biodegradable polymer-based poly(lactic-coglycolic acid) (PLGA) as a nanoparticle core and used DSPE-PEG as a shell-forming lipid. The
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presence of PEG on the outer surface may result in prolonged blood circulation and may improve
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the EPR.
Overall, we attempted to improve the therapeutic efficiency of anticancer drugs for treating
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osteosarcoma. To do this, we delivered a unique drug combination consisting of PTX and ETP
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by encapsulating the drugs in a novel lipid-polymer hybrid nanoparticle (Fig. 1). We hypothesized that PTX plus ETP could be innovative for synergistic therapy for osteosarcoma.
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We characterized the nanoparticles in terms of particle size, shape, and release kinetics. The synergistic anticancer efficacy of the drugs was evaluated using a cytotoxicity assay, an
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Materials and Methods
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apoptosis assay, and a caspase assay.
Materials
Poly (lactic-co-glycolic acid) (PLGA, molar ratio of D, L-lactic to glycolic acid, 50: 50) was purchased from Jinan Daigang Biotechnology, China. DSPE-PEG2000 was kindly gifted by Lipoid GmbH (Germany). Paclitaxel and Etoposide were purchased from Sigma-Aldrich, China. All other chemicals were of analytical grade and used without further purifications.
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Preparation of PTX/ETP-loaded lipid-polymer hybrid nanoparticles The LPN was prepared by emulsification-sonication method. Briefly, PTX (5 mg), ETP (5 mg),
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and PLGA (65 mg) was mixed in 10 ml of DMSO and allowed to stir for 10 min. Separately, 50 mg of DSPE-PEG and 2 mg of cholesterol was dissolved in water and sonicated for 15 min. The
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oil phase was dissolve in aqueous phase and ultrasonicated for 10 min. The mixture was then stirred for 2h until all the organic solvents were removed. The suspension was centrifuged to
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collect the drug-loaded nanoparticles. The free drugs present in the supernatant was removed and
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quantified using HPLC method. Hitachi L-400 HPLC was used and samples were injected into a C18 column (Sepax BR-C18, 5 m, 120 A˚, 4.6 × 150 mm).
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Particle size analysis
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The particle size, polydispersity index (PDI) and zeta potential was measured using dynamic
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light scattering (DLS) technique. ELS-Z (Photal, Japan) was used to measure the particle size at
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25°C. The samples were suitably diluted with distilled water and measured in triplicate. Morphology analysis
The particle morphology was determined using transmission electron microscopy (TEM) using H7600, Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV. The samples were suitably diluted and counterstained with 2% phosphotungistic acid and placed on a carbon coated copper grid in 400 meshes. The samples were dried and viewed under TEM. In vitro drug release Dialysis was used to evaluate drug release. Briefly, lyophilized samples containing 2 mg PTX/ETP equivalent were reconstituted in 1 mL distilled water and packed in a dialysis 6 Page 8 of 32
membrane (pore size, 3.5 kDa; Sigma-Aldrich), after which the ends were sealed. Then we placed the dialysis membrane in 30 mL phosphate buffered saline (PBS, pH 7.4) and acetate buffered saline (ABS, pH 5.0) at 37°C. The release study was started, and samples were
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withdrawn at a fixed time interval from 1 to 100 h. The samples were subjected to HPLC
mobile phase was varied and used in an alternative manner.
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Cellular uptake of NPs
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analysis to quantify the amount of each drug released at each time point. For different drugs, the
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The cellular uptake of nanoparticles was studied by confocal laser scanning microscope (CLSM). Rhodamine-B (Sigma-Aldrich, China) was used as a fluorescent probe. Briefly, cells were
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seeded in 6-well plate containing a cover glass chamber and incubated overnight. The cells were then exposed with rhodamine-B loaded NP and incubated for 2h. After cell incubation, cells
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were fixed with 4% paraformaldehyde. The cells were then stained with 4,6-diamidino-2-
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phenylindole (DAPI) to stain nuclei. The cells were observed through confocal laser scanning
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microscope (LSM 410, Zeiss, Jena, Germany). Cytotoxicity assay
MTT assay was performed to analyse the cytotoxicity potential of drugs [19,20]. The principle is that the yellow MTT is reduced by mitochondrial succinate dehydrogenase after entering into the live cells. For this purpose, 10000 cells/well was seeded in a 96-well plate and incubated for 24h. After 24h, old media was replaced with fresh media containing blank PLN, free PTX, ETP, and PE-LPN in a concentration-dependent manner ranging from 0.01 to 10 µg/ml. The formulations were incubated for 24h [21-23]. The cells were treated with MTT solution (10 µl, 5 mg/ml) and further incubated for 4h. The MTT media was removed carefully and added with 200 µL of 7 Page 9 of 32
DMSO to each well to solubilize the formazan salts. The absorbance of the solution was measured using a microplate reader (Beckman Coulter DTX880, USA) at 570 nm [16].
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Apoptosis analysis Annexin-V/PI staining using flow cytometer was performed for apoptosis analysis. The MG63
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cancer cells at a seeding density of 3×105 cells/well was seeded in a 6-well plate and incubated
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for 24h. The cells were exposed with free PTX, ETP, PTX/ETP and PE-LPN and incubated for 24h. Next day, cells were trypsinized, washed, and the pellet was treated with Annexin-V FITC
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(2 µl) and PI (2 µl) and incubated for 15 min. The cells were then measured by flow cytometer
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(FACS Calibur, BD Biosciences). Caspase 3 & 8 Activity
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Colorimetric assay kits (Sigma-Aldrich) were used to analyze caspase-3 and -8 activity. Briefly,
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1 × 106 cells were seeded in a 6-well plate and allowed to attach overnight. The next day, free
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PTX, ETP, PTX/ETP, and PE-LPN were exposed to cells and incubated for 24 h. Cells were extracted and lysed using a lysis buffer for 10 min (in ice). The lysate was collected by high centrifugation of lysed cells, and the supernatant was used for caspase analysis. A total of 20 µL cell lysate was mixed with p-nitroaniline (pNA)-conjugated substrate for caspase-3 (AcDEVDpNA) and -8 (Ac-IETD-pNA) and incubated for 1 h at 37°C. A microplate reader was used to detect fluorescence. The amount of released pNA was measured based on the absorbance values at 405 nm. Caspase activity was expressed as fold increases compared to an untreated cell control.
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Antitumor efficacy study and immunohistochemical analysis The animal study was approved by the Institutional Animal Ethics Committee, China. The
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antitumor efficacy study was performed in MG-63 cancer cell bearing xenograft tumor model. The tumor was developed on the right flank of nude mice and allowed to grow for 2 weeks from
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the time of injection. The mice were randomly divided into 5 groups with 6 mice in each group. The mice were administered with respective formulations with a fixed dose of 5 mg/kg (3 times
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during 10 days). The tumor volume was measured using Vernier caliper. At the end of study,
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tumors were removed and subjected to Ki67 analysis to evaluate the apoptosis cells.
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Statistical analysis
The data obtained were expressed in terms of “mean±standard deviation” values. The data were
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also subjected to one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test.
Results
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A value of p <0.05 was considered as significant.
Characterization of PTX/ETP-loaded lipid-polymer hybrid nanoparticles The particle diameter of PE-LPN was in the range of ~190 nm, with an excellent polydispersity index (PDI ~0.15), which indicates the success of the formulation technique (Fig. 2a). The surface charge of PE-LPN was –22.8 mV, which indicates its excellent physical stability. The surface morphology of NP was evaluated based on TEM (Fig. 2b). Perfect spherically shaped particles were observed using TEM. The size observed in TEM was similar to that observed in
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DLS. The drug entrapment efficiency (EE) for PTX was ~92%, whereas ETP showed ~90% with active drug loading of 8–10%.
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In vitro drug release Drug release was evaluated using dialysis. As shown in Figure 2c, both drugs were released in a
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well-controlled manner throughout the study period. Although different release patterns were
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observed for the two drugs, an initial burst in release was not observed, which indicates that small molecules were present stably in the core of the LPN. For example, ~20% of PTX was
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released from LPN, whereas ~15% of ETP was released from the carrier within 24 h. After 96 h,
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~85% of PTX was released compared to ~65% for ETP. Cellular uptake
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The cellular uptake of nanoparticles in cancer cells was investigated using a confocal laser
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scanning microscope (CLSM). Nuclei were stained with DAPI, and rhodamine-B was used as a
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fluorescent probe. As seen in Figure 3, red fluorescence accumulated predominantly in the cell cytoplasm, which suggests that typical endocytosis-mediated cellular entry bypasses the p-gp efflux pump. The colocalization of nanoparticles in the endo/lysosomal region clearly indicates that the carrier degrades in this region and the released drug travels to the nucleus for its pharmacological actions. The small particle size and hydrophobic nature of NPs contributed to the high internalization efficiency [24]. Cytotoxicity study against MG63 osteosarcoma cells The biocompatibility of nanoparticles is an important concern when these nanoparticles are intended for clinical applications. We evaluated the biocompatibility profiles of blank
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nanoparticles. Based on our results, relative cell viability from 101% to 88% was observed when exposed at concentrations of 0.1 to 500 µg/mL (Fig. 4). The results clearly suggest that the nanoparticles or degradation products did not affect cancer cell growth. This supports the good
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biocompatible profile of LPN and its potential use as a biomaterial for drug delivery. We next evaluated the cytotoxicity of individual drugs and combined drugs/NPs in MG63 cancer cells
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(Fig. 5). Free PTX, ETP, PTX/ETP, and PE-LPN showed typical concentration-dependent
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cytotoxic effects in MG63 cancer cells. Note that PTX showed a greater cytotoxic effect than ETP. The cocktail combination of PTX/ETP induced a significantly greater cytotoxic effect in
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cancer cells. Most notable is that nanoparticle-based PE-LPN showed the greatest anticancer effect of all of the formulations. PTX, ETP, and PTX/ETP showed cell viabilities of 65%, 82%,
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and 57%, respectively.
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Apoptosis assay
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As seen, PTX and ETP induced the apoptosis of cancer cells while cocktail combination of
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PTX/ETP induced a higher cancer cell apoptosis (Fig. 6). Approximately, ~26% of cells underwent apoptosis compared to only ~15% by individual drugs. Importantly, PE-LPN exhibited a significantly higher apoptosis of cancer cells (~45%) compared to that of any other groups.
Caspase-3 & -8 analyses
The effects of individual formulations on apoptosis were further confirmed based on caspase-3 and -8 analysis (Fig. 7). Cells were treated with the respective formulations, and caspase levels were estimated as part of the evaluation of therapeutic efficacy. Consistent with the cell apoptosis assay, PE-LPN showed significantly greater caspase-3 and -8 activity in cancer cells. 11 Page 13 of 32
The significantly greater activity of caspases was attributed to the encapsulation of the drugs in the nanoparticulate system and the controlled release of the drugs into the intracellular
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environment. In vivo antitumor efficacy study
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We evaluated in vivo antitumor efficacy in an MG63 cancer cell-bearing xenograft tumor model.
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Tumor-bearing mice were divided equally into five groups: control, PTX, ETP, PTX/ETP, and PE-LPN. Tumors grew exponentially in the untreated mice, whereas administration of free drugs
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controlled tumor growth to an extent. The cocktail mixture of free drugs controlled the progression of the tumors to some extent but failed to stop their growth completely. PE-LPN was
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very effective at controlling the growth of tumor cells, with a final tumor volume of ~400 mm3 compared to ~1500 mm3 in the control. The group treated with PE-LPN consistently showed
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Discussion
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a single drug.
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significantly fewer Ki-67-positive cells (less than 25%) than the groups treated with PTX/ETP or
The standard treatment protocol for osteosarcoma is surgery and conventional chemotherapy. However, single-drug therapy is ineffective because of drug resistance mechanisms coupled with the complex microenvironment of tumor tissues. Combination therapy is effective for treating drug-resistant cancers as it increases pharmacological activity and decreases side effects. In this study, we used the new combination of paclitaxel (PTX) and etoposide (ETP) to treat osteosarcoma. PTX stabilizes microtubules and arrests cancer cells in the G2/M phase, inducing apoptosis and cell death. ETP induces cancer cell death by stabilizing the covalent enzyme–DNA complex that is important for the topoisomerase II cycle. However, the therapeutic efficacy of 12 Page 14 of 32
multiple drugs can be hampered by poor pharmacokinetic performance, biodistribution, and membrane transport properties of the drugs. Therefore, we developed a novel lipid-polymer hybrid nanoparticle (LPN) to improve the anticancer efficacy of chemotherapeutic combinations
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and used PLGA as a nanoparticle core and DSPE-PEG as a shell-forming lipid. The presence of
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PEG on the outer surface extends blood circulation and may improve the EPR (Fig. 1).
The particle diameter of PE-LPN was in the range of ~190 nm, with an excellent polydispersity
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index (PDI~0.15). A particle less than <200 nm in diameter is expected to increase cellular
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internalization and improve intracellular concentrations of both drugs. Nanosized particles can prolong blood circulation and enhance accumulation in tumor tissues through the EPR effect. In
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addition, small particles can diminish the clearance of loaded drugs from systemic circulation [25]. The zeta potential plays an important role in physical stability and biological applications
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of nanoparticles (PE-LPN, –22.8 mV).
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Both drugs were released in a well-controlled manner throughout the study period. Differences in
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the release pattern may be associated with differences in the physicochemical characteristics of PTX (Log P ~3.96) and ETP (Log P ~1.16). These results indicate that LPN may encapsulate both drugs efficiently and control the release kinetics in the medium. According to our results, NPs can take advantage of the acidic pH of the tumor environment and release their payload at the tumor site, and systemic side effects will decrease during cancer treatment. This release pattern is particularly important to maintaining improved drug delivery in the tumor microenvironment and intracellular compartments. Furthermore, a sequential pattern of drug release can induce cell apoptosis in cancer tissues.
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The PE-LPN showed a low cell viability of 45%, which indicates the excellent cytotoxic effects of the combination of chemotherapeutic drugs. The greater anticancer effects of PE-LPN were due to increased cellular internalization and controlled drug release in the intracellular
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environment. The greater accumulation of drugs in the cancer cells resulted in an increased anticancer effect in the osteosarcoma cells. To increase our understanding of the effects of
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different micelle formulations on cell growth, we calculated the IC50 value (the concentration at
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which 50% of cells are killed). The IC50 values of free PTX, ETP, PTX/ETP, and PE-LPN were 3.86 µg/mL, 7.89 µg/mL, 1.98 µg/mL, and 0.74 µg/mL, respectively. According to these IC50
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values, combinations of nanoparticles show superior performance in cancer cells. Furthermore, combinations of nanoparticles are more effective than cocktail free drugs at killing cancer cells.
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The combination of anticancer drugs resulted in an increased therapeutic efficacy and target
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selectivity. The administration of small molecules that alter signaling pathways can delay cancer
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cell proliferation and exhibit an enhanced cell-killing effect [26,27].
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Apoptosis, programmed cell death marked by numerous biochemical and morphological changes, plays an important role in tissue homeostasis. Loss of membrane potential and the presence of phosphatidylserine (PS) are standard parameters in analyses of apoptosis. An Annexin/PI staining-based apoptosis assay was performed in MG63 cancer cells. The scatter plot could be divided into four quadrants: viable cells (Annexin − ve, PI − ve), represented by the lower left quadrant (Q3); early apoptotic cells (Annexin + ve, PI − ve), represented by the lower right quadrant (Q4); late apoptotic cells (Annexin + ve, PI + ve), represented by the upper right quadrant (Q2); and necrotic cells (PI + ve), represented by the upper left quadrant (Q1). Consistent with the cytotoxicity assay, the apoptosis results showed increased cancer cell apoptosis upon incubation with combinations of drugs. In addition, cocktail drugs showed a 14 Page 16 of 32
synergistic effect; however, the therapeutic effect was greater when drugs were encapsulated in the nanoparticulate system because of the controlled release of the drug in a sequential pattern.
the drugs resulted in higher apoptosis effects of PE-LPN [18,22].
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Furthermore, the increased internalization efficiency of the NP system and modifiable release of
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After the in vitro analysis, we studied antitumor efficacy in mice with MG63 cancer cell tumors. As seen in Figure 8a, free drugs had a limited effect on controlling tumor progression, with
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tumor volumes around ~1000 mm3. The cocktail mixture of free drugs controlled tumor
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progression to some extent but failed to stop growth completely. As expected, PE-LPN had a significant tumor regression effect and 2-fold superior efficacy compared to free drugs. The final
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tumor volume of PE-LPN was ~450 mm3. The superior antitumor efficacy of PE-LPN is attributed to its nanosized and controlled drug release kinetics. The nanoparticles internalize
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preferentially and show a high intracellular concentration compared to cocktail combinations
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[10,18]. Moreover, the presence of PEG on the outer shell improved blood circulation, which
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may have resulted in enhanced tumor accumulation. In addition, to differentiate between proliferating cells remaining in the tumor tissue and dead cell populations, we performed a Ki-67 cell proliferation assay based on immunohistochemical staining of the tumor sections (Fig. 8b, 8c). Ki-67 is a proliferative cell nuclear antigen present during all active cell phases, including G1, S, and G2, and is commonly used as a cell proliferation marker. As shown in Figure 8, a high Ki-67 positivity rate was observed in the control group, which indicates active cell proliferation. By contrast, the group treated with PE-LPN showed significantly fewer Ki-67positive cells (less than 25%) than the groups treated with PTX/ETP or a single drug, which suggests less active cell proliferation and a considerably greater tumor growth inhibition effect [18]. 15 Page 17 of 32
Conclusions In summary, paclitaxel- and etoposide-loaded lipid-polymer hybrid nanoparticles (PE-LPN) were
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successfully prepared and evaluated for physicochemical and anticancer effects. Nanosized PELPN was obtained with a perfect spherical morphology. PE-LPN exhibited the controlled release
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of two drugs in a sequential manner. The nanoparticles showed typical endocytosis-mediated cellular uptake in cancer cells. The ratiometric combination of PTX and ETP was significantly
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more cytotoxic than the individual drugs. Note that a superior cytotoxic effect was observed for
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dual drug–loaded PE-LPN compared to a cocktail combination at a much lower dose. Similarly, PE-LPN showed significantly higher apoptosis of cancer cells (~45%) compared to other groups
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with greater caspase-3 and -8 activity. Note that PE-LPN showed a remarkable tumor regression effect and exhibited 2-fold superior efficacy compared to free drugs. The group treated with PE-
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LPN showed significantly fewer Ki-67-positive cells (less than 25%) than the groups treated
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with PTX/ETP or a single drug, which suggests less active cell proliferation and a considerably
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greater tumor growth inhibition effect. The results show that combining drugs can significantly improve the therapeutic properties of chemotherapeutic drugs. The therapeutic efficacy of ETP and PTX (a powerful anticancer drug) for treating osteosarcoma is improved when the drugs are combined in a polymer-lipid hybrid nanoparticle system.
Acknowledgement This study was supported from the grant of Luoyang Orthopedic Hospital of Henan Province, China.
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selectivity. Nat. Biotechnol. 27 (2009) 659–666. [16]. T. Ramasamy, H.B. Ruttala, B.G. Kanu, B.K. Poudel, H.G. Choi, C.S. Yong, J.O. Kim. Smart chemistry-based nanosized drug delivery systems for systemic applications: A comprehensive review. Journal of Controlled Release 2017, pii: S0168-3659(17)30559-X. doi: 10.1016/j.jconrel.2017.04.043.
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[17]. L. Zhang et al. Folate-modified lipid-polymer hybrid nanoparticles for targeted paclitaxel delivery. Int. J. Nanomedicine. 10 (2015) 2101-2114. [18]. T. Ramasamy et al. Engineering of a lipid-polymer nanoarchitectural platform for highly
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effective combination therapy of doxorubicin and irinotecan. Chem. Commun (Camb). 51 (2015) 5758-5761
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[19]. S. Khan, A.A. Ansari, A.A. Khan, M. Abdulla, O. Al-Obaid, R. Ahmad. In vitro evaluation
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of cytotoxicity, possible alteration of apoptotic regulatory proteins, and antibacterial activity of
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synthesized copper oxide nanoparticles. Colloids Surf B Biointerfaces. 153 (2017) 320-326. [20]. S. Khan, A.A. Ansari, A.A. Khan, R. Ahmad, O. Al-Obaid, W. Al-Kattan. In vitro
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evaluation of anticancer and antibacterial activities of cobalt oxide nanoparticles. J Biol Inorg Chem 20 (2015) 1319.
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[21]. Shahanavaj et al. Evaluation of in vitro cytotoxicity, biocompatibility, and changes in the
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expression of apoptosis regulatory proteins induced by cerium oxide nanocrystals Science and
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Technology of Advanced Materials 18 (2017) 364-373. [22]. S. Khan, A.A. Ansari, A.A. Khan et al. In vitro evaluation of anticancer and biological activities of synthesized manganese oxide nanoparticles. Med Chem Commun. 7 (2016) 1647 [23]. Khan et al..Design, synthesis and in vitro evaluation of anticancer and antibacterial potential of surface modified Tb(OH)3@SiO2 core-shell nanoparticles. RSC Adv. 6 (2016) 18667. [24]. S. Shishodia, H.M. Amin, R. Lai, B.B. Aggarwal, Biochem. Pharmacol. 70 (2005) 700– 713.
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[25].
T.
paclitaxel
Shimada,
M.
Ueda,
incorporated
into
H.
Jinno.
Development
EGF-conjugated
of
targeted
nanoparticles.
therapy
with
Anticancer
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29 (2009) 1009–1014. Anitha,
Combinatorial thiolated
N.
Deepa,
anticancer
chitosan
K.P.
Chennazhi,
effects
nanoparticles
of
V.K.
curcumin
towards
colon
Lakshmanan,
R.
Jayakumar.
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A.
and
5-fluorouracil
cancer
treatment.
loaded
Biochim.
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Biophys. Acta. 1840 (2014) 2730–2743.
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[26].
[27]. J.S. Park, T.H. Han, K.Y. Lee, S.S. Han, J.J. Hwang, D.H. Moon, et al. N-acetyl
an
histidine conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery
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te
d
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of drugs: endocytosis, exocytosis and drug release. J. Control. Release. 115 (2006) 37–44
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Figure captions Figure 1: Schematic illustration of preparation of paclitaxel and etoposide-loaded lipid-polymer
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hybrid nanoparticles Figure 2: (a) Particle size distribution of PE-LPN (b) transmission electron microscope (TEM)
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image of PE-LPN. (c) In vitro drug release profile of PTX and ETP from PE-LPN in PBS buffer.
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The release study was carried out in PBS at 37°C. Results are expressed as mean±S.D (n=3). Figure 3: In vitro cellular uptake of PE-LPN formulations in MG63 cells. CLSM analysis of
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MG63 cells after treatment with PE-LPN after 2h incubation (DAPI dye: blue, and rhodamine-B:
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red).
Figure 4: In vitro cytotoxicity of blank nanoparticles at various concentrations against MG63.
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The cells were incubated with blank NP for 24 h and the cytotoxicity assay was performed by
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MTT reagent. Results are expressed as mean±S.D (n=8).
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Figure 5: In vitro cytotoxicity of free PTX, free ETP, free PTX/ETP, and PE-LPN against MG63. The cytotoxicity assay was performed by MTT assay. **p<0.01 is the statistical difference between PE-LPN and PTX and ETP, respectively. Results are expressed as mean±S.D (n=8). Statistical differences were tested by one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test
Figure 6: Flow cytometer analysis of cell apoptosis using annexinV-FITC and PI staining. The MG63 cells were exposed with free PTX, free ETP, free PTX/ETP, and PE-LPN at a concentration of 1000 ng/ml and incubated for 24 h. ***p<0.001 is the statistical difference between PE-LPN and PTX and ETP, respectively. Results are expressed as mean±S.D (n=3).
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Statistical differences were tested by one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test
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Figure 7: Caspase-3 and 8 activities were measured as a second parameter of apoptotic cell death in a MG63 cancer cells. Significant increase in apoptosis was observed when they were treated
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with combinational nanoparticles. **p<0.01 is the statistical difference between PE-LPN and PTX and ETP, respectively. Results are expressed as mean±S.D (n=3). Statistical differences
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were tested by one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test
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Figure 8: (a) Antitumor efficacy study in terms of tumor volume in MG63 xenograft tumor
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Results are expressed as mean±S.D (n=6).
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model (b) immunohistochemical analysis of tumor mass for Ki67+ (C) histogram of Ki67+ cells.
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S0927-7765(17)30557-X http://dx.doi.org/doi:10.1016/j.colsurfb.2017.08.042 COLSUB 8798
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-12-2016 21-8-2017 23-8-2017
Please cite this article as: R. Duan, C. Li, F. Wang, J.-C. Yangi, Polymer-lipid hybrid nanoparticles-based paclitaxel and etoposide combinations for the synergistic anticancer efficacy in osteosarcoma, Colloids and Surfaces B: Biointerfaces (2017), http://dx.doi.org/10.1016/j.colsurfb.2017.08.042 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.
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1. Paclitaxel and etoposide-loaded lipid-polymer hybrid nanoparticles was prepared for
enhanced efficacy in osteosarcoma 2. PE-LPN exhibited a remarkably higher apoptosis cell death of cancer cells
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3. PE-LPN showed a remarkable tumor regression effect and exhibited a 2-fold superior
efficacy than free drugs
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4. Combination of drugs in a LPN carrier could greatly improve the therapeutic property of
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chemotherapeutic drugs
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Polymer-lipid hybrid nanoparticles-based paclitaxel and etoposide combinations for the synergistic anticancer efficacy in osteosarcoma
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Rui Duan1#, Caiyan Li2#, Fan Wang3*, Jin-Chu Yangi4*,
Department of oncology, The first People’s Hospital of Jingmen, Jingmen, Hubei 448000, China
2
Department of Clinical laboratory, The second People’s Hospital of Jingmen, Jingmen, Hubei
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1
3
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448000, China
Department of orthopaedics, The first People’s Hospital of Jingmen, Jingmen, Hubei 448000,
China
Department of hand surgery, Luoyang Orthopedic Hospital of Henan Province, Henan, 471002,
an
4
China
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#These two authors contribute to this work equally
Corresponding author:
Dr. Jin-Chu Yangi, Department of hand surgery, Luoyang Orthopedic Hospital of Henan Province, No.233, Qiming South Road, Luoyang, 471002, Henan, P.R.China Tel&Fax: 0086-379-63546996 Email: [email protected]
Dr. Fan Wang, Department of orthopaedics, Jingmen No.1 People’s Hospital, No.67 of Xiangshan Road, Jingmen, Hubei 448000, China Tel/Fax: 0086- 07242305966 [email protected] 1 Page 3 of 32
Abstract In this study, paclitaxel and etoposide-loaded lipid-polymer hybrid nanoparticles (PE-LPN) was
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successful prepared and evaluated for physicochemical and anticancer effect. Nanosized PE-LPN was obtained with a perfect spherical morphology. PE-LPN exhibited a controlled release of two
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drugs in a sequential manner. The nanoparticles exhibited a typical endocytosis-mediated cellular uptake in cancer cells. The ratiometric combination of paclitaxel (PTX) and Etoposide (ETP)
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were significantly more cytotoxic than individual drugs. Importantly, superior cytotoxic effect
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was observed for dual-drug-loaded PE-LPN than cocktail combination at a much lower dose. Similarly, PE-LPN exhibited a significantly higher apoptosis of cancer cells (~45%) compared to
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that of any other groups with higher caspase-3 & -8 activity. Importantly, PE-LPN showed a remarkable tumor regression effect and exhibited a 2-fold superior efficacy than free drugs. PE-
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LPN treated group showed significantly less Ki-67 positive cells (less than 25%) than PTX/ETP
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and single drug treated groups, suggesting less active cell proliferation and a considerably higher
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tumour growth inhibition effect. The results collectively showed that combination of drugs could greatly improve the therapeutic property of chemotherapeutic drugs. By combining ETP with PTX (a powerful anticancer drug) in a polymer–lipid hybrid nanoparticle system, therapeutic efficacy could be improved in osteosarcoma treatments.
Keywords Osteosarcoma, paclitaxel, etoposide, apoptosis, nanoparticles
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Introduction Osteosarcoma is one of the common forms of bone cancer affecting adult people aging between
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10 and 24 years [1,2]. Like the osteoblasts in normal bone, the cells that form this cancer make bone matrix. But the bone matrix of an osteosarcoma is not as strong as that of normal bones.
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Osteosarcoma is a bone cancer that typically develops in the shinbone (tibia) near the knee, the thighbone (femur) near the knee, or the upper arm bone (humerus) near the shoulder.
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Osteosarcoma tends to develop during growth spurts in early adolescence. This may be because
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the risk of tumors increases during this period of rapid bone growth. It accounts for nearly 60% of all bone cancers in Children and adults. It is diagnosed mostly in first 2 decades of their life
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term. It accounts for nearly 60% of all bone cancers in children and adults and is typically diagnosed in the first two decades of life. Despite significant advancements in the diagnosis and
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treatment of osteosarcoma, the 5-year survival rate is less than 65%, and metastatic cancer
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accounts for less than 20% of cases [3]. Surgery and conventional chemotherapy are the standard
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treatment for this disease, and the cure rate increases when chemotherapy is combined with surgery. Several classes of single anticancer drugs have been indicated for the treatment of osteosarcoma; however, failure of the chemotherapy regimen is a major factor in unsuccessful treatment [4,5]. Failure mainly results from drug resistance in patients with advanced-stage cancer. The complex microenvironment of cancer cells coupled with drug resistance contributes to the failure of chemotherapy based on a single drug [6,7]. Therefore, combination chemotherapy has been used to improve the therapeutic efficacy of drugs while simultaneously reducing drug-related side effects. Combination therapy is effective for treating drug-resistant cancers, improving therapeutic efficacy, and minimizing adverse effects [8]. Using more than two drugs that target different 3 Page 5 of 32
pharmacological pathways could alter genetic barriers, including cancer cell mutations and adaptations, and suppress drug efflux receptors while acting in a synergistic manner. Therefore, the use of a combination of anticancer drugs could result in greater therapeutic efficacy and
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target selectivity [9,10]. Here we investigated the suitability of a combination of paclitaxel (PTX) and etoposide (ETP) for treating osteosarcoma. PTX acts by stabilizing microtubules and
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blocking cancer cells at the G2/M phase, provoking cell apoptosis. In addition, PTX can enhance
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expression of nuclear factor-B (NF-B), a transcription factor that regulates various physiological processes, including inflammation, differentiation, and apoptosis [11,12]. ETP stabilizes covalent
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enzyme–DNA complexes that play important roles in the catalytic cycle of topoisomerase II. The accumulation of cleaved enzyme complex leads to a permanent break in the DNA strand,
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resulting in chromosomal translocation and eventual cell death [13,14].
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The anticancer efficacy of a combination of drugs could be severely hindered by the acute and
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chronic toxicity of the individual drugs. In addition, the therapeutic efficacy of two drugs could
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be hampered by poor pharmacokinetic performance, biodistribution, and membrane transport properties, which may lead to poor internalization into tumor tissues [15]. Therefore, it is important to improve the physicochemical properties and anticancer efficacy of dual drugs while limiting their side effects. In this regard, nanoparticles may be used to circumvent problems associated with drug delivery and improve pharmacokinetic parameters [16]. In particular, lipidpolymer hybrid nanoparticles (LPN) offer many attractive features for improving the anticancer efficacy of chemotherapeutic combinations. LPN offers high systemic stability, high drug loading, high protection of encapsulated compounds, and prolonged blood circulation. Nanosized particles will preferentially accumulate in tumor tissues via the enhanced permeation and retention (EPR) effect [17,18]. In addition, nanosized carriers consisting of naturally derived 4 Page 6 of 32
materials evade the reticuloendothelial system (RES) and inhibit systemic clearance, thereby prolonging drug circulation. Therefore, we used a biodegradable polymer-based poly(lactic-coglycolic acid) (PLGA) as a nanoparticle core and used DSPE-PEG as a shell-forming lipid. The
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presence of PEG on the outer surface may result in prolonged blood circulation and may improve
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the EPR.
Overall, we attempted to improve the therapeutic efficiency of anticancer drugs for treating
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osteosarcoma. To do this, we delivered a unique drug combination consisting of PTX and ETP
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by encapsulating the drugs in a novel lipid-polymer hybrid nanoparticle (Fig. 1). We hypothesized that PTX plus ETP could be innovative for synergistic therapy for osteosarcoma.
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We characterized the nanoparticles in terms of particle size, shape, and release kinetics. The synergistic anticancer efficacy of the drugs was evaluated using a cytotoxicity assay, an
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Materials and Methods
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apoptosis assay, and a caspase assay.
Materials
Poly (lactic-co-glycolic acid) (PLGA, molar ratio of D, L-lactic to glycolic acid, 50: 50) was purchased from Jinan Daigang Biotechnology, China. DSPE-PEG2000 was kindly gifted by Lipoid GmbH (Germany). Paclitaxel and Etoposide were purchased from Sigma-Aldrich, China. All other chemicals were of analytical grade and used without further purifications.
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Preparation of PTX/ETP-loaded lipid-polymer hybrid nanoparticles The LPN was prepared by emulsification-sonication method. Briefly, PTX (5 mg), ETP (5 mg),
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and PLGA (65 mg) was mixed in 10 ml of DMSO and allowed to stir for 10 min. Separately, 50 mg of DSPE-PEG and 2 mg of cholesterol was dissolved in water and sonicated for 15 min. The
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oil phase was dissolve in aqueous phase and ultrasonicated for 10 min. The mixture was then stirred for 2h until all the organic solvents were removed. The suspension was centrifuged to
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collect the drug-loaded nanoparticles. The free drugs present in the supernatant was removed and
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quantified using HPLC method. Hitachi L-400 HPLC was used and samples were injected into a C18 column (Sepax BR-C18, 5 m, 120 A˚, 4.6 × 150 mm).
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Particle size analysis
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The particle size, polydispersity index (PDI) and zeta potential was measured using dynamic
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light scattering (DLS) technique. ELS-Z (Photal, Japan) was used to measure the particle size at
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25°C. The samples were suitably diluted with distilled water and measured in triplicate. Morphology analysis
The particle morphology was determined using transmission electron microscopy (TEM) using H7600, Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV. The samples were suitably diluted and counterstained with 2% phosphotungistic acid and placed on a carbon coated copper grid in 400 meshes. The samples were dried and viewed under TEM. In vitro drug release Dialysis was used to evaluate drug release. Briefly, lyophilized samples containing 2 mg PTX/ETP equivalent were reconstituted in 1 mL distilled water and packed in a dialysis 6 Page 8 of 32
membrane (pore size, 3.5 kDa; Sigma-Aldrich), after which the ends were sealed. Then we placed the dialysis membrane in 30 mL phosphate buffered saline (PBS, pH 7.4) and acetate buffered saline (ABS, pH 5.0) at 37°C. The release study was started, and samples were
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withdrawn at a fixed time interval from 1 to 100 h. The samples were subjected to HPLC
mobile phase was varied and used in an alternative manner.
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Cellular uptake of NPs
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analysis to quantify the amount of each drug released at each time point. For different drugs, the
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The cellular uptake of nanoparticles was studied by confocal laser scanning microscope (CLSM). Rhodamine-B (Sigma-Aldrich, China) was used as a fluorescent probe. Briefly, cells were
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seeded in 6-well plate containing a cover glass chamber and incubated overnight. The cells were then exposed with rhodamine-B loaded NP and incubated for 2h. After cell incubation, cells
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were fixed with 4% paraformaldehyde. The cells were then stained with 4,6-diamidino-2-
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phenylindole (DAPI) to stain nuclei. The cells were observed through confocal laser scanning
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microscope (LSM 410, Zeiss, Jena, Germany). Cytotoxicity assay
MTT assay was performed to analyse the cytotoxicity potential of drugs [19,20]. The principle is that the yellow MTT is reduced by mitochondrial succinate dehydrogenase after entering into the live cells. For this purpose, 10000 cells/well was seeded in a 96-well plate and incubated for 24h. After 24h, old media was replaced with fresh media containing blank PLN, free PTX, ETP, and PE-LPN in a concentration-dependent manner ranging from 0.01 to 10 µg/ml. The formulations were incubated for 24h [21-23]. The cells were treated with MTT solution (10 µl, 5 mg/ml) and further incubated for 4h. The MTT media was removed carefully and added with 200 µL of 7 Page 9 of 32
DMSO to each well to solubilize the formazan salts. The absorbance of the solution was measured using a microplate reader (Beckman Coulter DTX880, USA) at 570 nm [16].
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Apoptosis analysis Annexin-V/PI staining using flow cytometer was performed for apoptosis analysis. The MG63
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cancer cells at a seeding density of 3×105 cells/well was seeded in a 6-well plate and incubated
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for 24h. The cells were exposed with free PTX, ETP, PTX/ETP and PE-LPN and incubated for 24h. Next day, cells were trypsinized, washed, and the pellet was treated with Annexin-V FITC
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(2 µl) and PI (2 µl) and incubated for 15 min. The cells were then measured by flow cytometer
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(FACS Calibur, BD Biosciences). Caspase 3 & 8 Activity
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Colorimetric assay kits (Sigma-Aldrich) were used to analyze caspase-3 and -8 activity. Briefly,
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1 × 106 cells were seeded in a 6-well plate and allowed to attach overnight. The next day, free
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PTX, ETP, PTX/ETP, and PE-LPN were exposed to cells and incubated for 24 h. Cells were extracted and lysed using a lysis buffer for 10 min (in ice). The lysate was collected by high centrifugation of lysed cells, and the supernatant was used for caspase analysis. A total of 20 µL cell lysate was mixed with p-nitroaniline (pNA)-conjugated substrate for caspase-3 (AcDEVDpNA) and -8 (Ac-IETD-pNA) and incubated for 1 h at 37°C. A microplate reader was used to detect fluorescence. The amount of released pNA was measured based on the absorbance values at 405 nm. Caspase activity was expressed as fold increases compared to an untreated cell control.
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Antitumor efficacy study and immunohistochemical analysis The animal study was approved by the Institutional Animal Ethics Committee, China. The
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antitumor efficacy study was performed in MG-63 cancer cell bearing xenograft tumor model. The tumor was developed on the right flank of nude mice and allowed to grow for 2 weeks from
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the time of injection. The mice were randomly divided into 5 groups with 6 mice in each group. The mice were administered with respective formulations with a fixed dose of 5 mg/kg (3 times
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during 10 days). The tumor volume was measured using Vernier caliper. At the end of study,
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tumors were removed and subjected to Ki67 analysis to evaluate the apoptosis cells.
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Statistical analysis
The data obtained were expressed in terms of “mean±standard deviation” values. The data were
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also subjected to one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test.
Results
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A value of p <0.05 was considered as significant.
Characterization of PTX/ETP-loaded lipid-polymer hybrid nanoparticles The particle diameter of PE-LPN was in the range of ~190 nm, with an excellent polydispersity index (PDI ~0.15), which indicates the success of the formulation technique (Fig. 2a). The surface charge of PE-LPN was –22.8 mV, which indicates its excellent physical stability. The surface morphology of NP was evaluated based on TEM (Fig. 2b). Perfect spherically shaped particles were observed using TEM. The size observed in TEM was similar to that observed in
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DLS. The drug entrapment efficiency (EE) for PTX was ~92%, whereas ETP showed ~90% with active drug loading of 8–10%.
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In vitro drug release Drug release was evaluated using dialysis. As shown in Figure 2c, both drugs were released in a
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well-controlled manner throughout the study period. Although different release patterns were
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observed for the two drugs, an initial burst in release was not observed, which indicates that small molecules were present stably in the core of the LPN. For example, ~20% of PTX was
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released from LPN, whereas ~15% of ETP was released from the carrier within 24 h. After 96 h,
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~85% of PTX was released compared to ~65% for ETP. Cellular uptake
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The cellular uptake of nanoparticles in cancer cells was investigated using a confocal laser
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scanning microscope (CLSM). Nuclei were stained with DAPI, and rhodamine-B was used as a
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fluorescent probe. As seen in Figure 3, red fluorescence accumulated predominantly in the cell cytoplasm, which suggests that typical endocytosis-mediated cellular entry bypasses the p-gp efflux pump. The colocalization of nanoparticles in the endo/lysosomal region clearly indicates that the carrier degrades in this region and the released drug travels to the nucleus for its pharmacological actions. The small particle size and hydrophobic nature of NPs contributed to the high internalization efficiency [24]. Cytotoxicity study against MG63 osteosarcoma cells The biocompatibility of nanoparticles is an important concern when these nanoparticles are intended for clinical applications. We evaluated the biocompatibility profiles of blank
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nanoparticles. Based on our results, relative cell viability from 101% to 88% was observed when exposed at concentrations of 0.1 to 500 µg/mL (Fig. 4). The results clearly suggest that the nanoparticles or degradation products did not affect cancer cell growth. This supports the good
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biocompatible profile of LPN and its potential use as a biomaterial for drug delivery. We next evaluated the cytotoxicity of individual drugs and combined drugs/NPs in MG63 cancer cells
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(Fig. 5). Free PTX, ETP, PTX/ETP, and PE-LPN showed typical concentration-dependent
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cytotoxic effects in MG63 cancer cells. Note that PTX showed a greater cytotoxic effect than ETP. The cocktail combination of PTX/ETP induced a significantly greater cytotoxic effect in
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cancer cells. Most notable is that nanoparticle-based PE-LPN showed the greatest anticancer effect of all of the formulations. PTX, ETP, and PTX/ETP showed cell viabilities of 65%, 82%,
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and 57%, respectively.
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Apoptosis assay
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As seen, PTX and ETP induced the apoptosis of cancer cells while cocktail combination of
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PTX/ETP induced a higher cancer cell apoptosis (Fig. 6). Approximately, ~26% of cells underwent apoptosis compared to only ~15% by individual drugs. Importantly, PE-LPN exhibited a significantly higher apoptosis of cancer cells (~45%) compared to that of any other groups.
Caspase-3 & -8 analyses
The effects of individual formulations on apoptosis were further confirmed based on caspase-3 and -8 analysis (Fig. 7). Cells were treated with the respective formulations, and caspase levels were estimated as part of the evaluation of therapeutic efficacy. Consistent with the cell apoptosis assay, PE-LPN showed significantly greater caspase-3 and -8 activity in cancer cells. 11 Page 13 of 32
The significantly greater activity of caspases was attributed to the encapsulation of the drugs in the nanoparticulate system and the controlled release of the drugs into the intracellular
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environment. In vivo antitumor efficacy study
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We evaluated in vivo antitumor efficacy in an MG63 cancer cell-bearing xenograft tumor model.
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Tumor-bearing mice were divided equally into five groups: control, PTX, ETP, PTX/ETP, and PE-LPN. Tumors grew exponentially in the untreated mice, whereas administration of free drugs
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controlled tumor growth to an extent. The cocktail mixture of free drugs controlled the progression of the tumors to some extent but failed to stop their growth completely. PE-LPN was
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very effective at controlling the growth of tumor cells, with a final tumor volume of ~400 mm3 compared to ~1500 mm3 in the control. The group treated with PE-LPN consistently showed
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Discussion
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a single drug.
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significantly fewer Ki-67-positive cells (less than 25%) than the groups treated with PTX/ETP or
The standard treatment protocol for osteosarcoma is surgery and conventional chemotherapy. However, single-drug therapy is ineffective because of drug resistance mechanisms coupled with the complex microenvironment of tumor tissues. Combination therapy is effective for treating drug-resistant cancers as it increases pharmacological activity and decreases side effects. In this study, we used the new combination of paclitaxel (PTX) and etoposide (ETP) to treat osteosarcoma. PTX stabilizes microtubules and arrests cancer cells in the G2/M phase, inducing apoptosis and cell death. ETP induces cancer cell death by stabilizing the covalent enzyme–DNA complex that is important for the topoisomerase II cycle. However, the therapeutic efficacy of 12 Page 14 of 32
multiple drugs can be hampered by poor pharmacokinetic performance, biodistribution, and membrane transport properties of the drugs. Therefore, we developed a novel lipid-polymer hybrid nanoparticle (LPN) to improve the anticancer efficacy of chemotherapeutic combinations
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and used PLGA as a nanoparticle core and DSPE-PEG as a shell-forming lipid. The presence of
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PEG on the outer surface extends blood circulation and may improve the EPR (Fig. 1).
The particle diameter of PE-LPN was in the range of ~190 nm, with an excellent polydispersity
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index (PDI~0.15). A particle less than <200 nm in diameter is expected to increase cellular
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internalization and improve intracellular concentrations of both drugs. Nanosized particles can prolong blood circulation and enhance accumulation in tumor tissues through the EPR effect. In
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addition, small particles can diminish the clearance of loaded drugs from systemic circulation [25]. The zeta potential plays an important role in physical stability and biological applications
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of nanoparticles (PE-LPN, –22.8 mV).
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Both drugs were released in a well-controlled manner throughout the study period. Differences in
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the release pattern may be associated with differences in the physicochemical characteristics of PTX (Log P ~3.96) and ETP (Log P ~1.16). These results indicate that LPN may encapsulate both drugs efficiently and control the release kinetics in the medium. According to our results, NPs can take advantage of the acidic pH of the tumor environment and release their payload at the tumor site, and systemic side effects will decrease during cancer treatment. This release pattern is particularly important to maintaining improved drug delivery in the tumor microenvironment and intracellular compartments. Furthermore, a sequential pattern of drug release can induce cell apoptosis in cancer tissues.
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The PE-LPN showed a low cell viability of 45%, which indicates the excellent cytotoxic effects of the combination of chemotherapeutic drugs. The greater anticancer effects of PE-LPN were due to increased cellular internalization and controlled drug release in the intracellular
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environment. The greater accumulation of drugs in the cancer cells resulted in an increased anticancer effect in the osteosarcoma cells. To increase our understanding of the effects of
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different micelle formulations on cell growth, we calculated the IC50 value (the concentration at
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which 50% of cells are killed). The IC50 values of free PTX, ETP, PTX/ETP, and PE-LPN were 3.86 µg/mL, 7.89 µg/mL, 1.98 µg/mL, and 0.74 µg/mL, respectively. According to these IC50
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values, combinations of nanoparticles show superior performance in cancer cells. Furthermore, combinations of nanoparticles are more effective than cocktail free drugs at killing cancer cells.
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The combination of anticancer drugs resulted in an increased therapeutic efficacy and target
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selectivity. The administration of small molecules that alter signaling pathways can delay cancer
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cell proliferation and exhibit an enhanced cell-killing effect [26,27].
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Apoptosis, programmed cell death marked by numerous biochemical and morphological changes, plays an important role in tissue homeostasis. Loss of membrane potential and the presence of phosphatidylserine (PS) are standard parameters in analyses of apoptosis. An Annexin/PI staining-based apoptosis assay was performed in MG63 cancer cells. The scatter plot could be divided into four quadrants: viable cells (Annexin − ve, PI − ve), represented by the lower left quadrant (Q3); early apoptotic cells (Annexin + ve, PI − ve), represented by the lower right quadrant (Q4); late apoptotic cells (Annexin + ve, PI + ve), represented by the upper right quadrant (Q2); and necrotic cells (PI + ve), represented by the upper left quadrant (Q1). Consistent with the cytotoxicity assay, the apoptosis results showed increased cancer cell apoptosis upon incubation with combinations of drugs. In addition, cocktail drugs showed a 14 Page 16 of 32
synergistic effect; however, the therapeutic effect was greater when drugs were encapsulated in the nanoparticulate system because of the controlled release of the drug in a sequential pattern.
the drugs resulted in higher apoptosis effects of PE-LPN [18,22].
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Furthermore, the increased internalization efficiency of the NP system and modifiable release of
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After the in vitro analysis, we studied antitumor efficacy in mice with MG63 cancer cell tumors. As seen in Figure 8a, free drugs had a limited effect on controlling tumor progression, with
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tumor volumes around ~1000 mm3. The cocktail mixture of free drugs controlled tumor
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progression to some extent but failed to stop growth completely. As expected, PE-LPN had a significant tumor regression effect and 2-fold superior efficacy compared to free drugs. The final
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tumor volume of PE-LPN was ~450 mm3. The superior antitumor efficacy of PE-LPN is attributed to its nanosized and controlled drug release kinetics. The nanoparticles internalize
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preferentially and show a high intracellular concentration compared to cocktail combinations
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[10,18]. Moreover, the presence of PEG on the outer shell improved blood circulation, which
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may have resulted in enhanced tumor accumulation. In addition, to differentiate between proliferating cells remaining in the tumor tissue and dead cell populations, we performed a Ki-67 cell proliferation assay based on immunohistochemical staining of the tumor sections (Fig. 8b, 8c). Ki-67 is a proliferative cell nuclear antigen present during all active cell phases, including G1, S, and G2, and is commonly used as a cell proliferation marker. As shown in Figure 8, a high Ki-67 positivity rate was observed in the control group, which indicates active cell proliferation. By contrast, the group treated with PE-LPN showed significantly fewer Ki-67positive cells (less than 25%) than the groups treated with PTX/ETP or a single drug, which suggests less active cell proliferation and a considerably greater tumor growth inhibition effect [18]. 15 Page 17 of 32
Conclusions In summary, paclitaxel- and etoposide-loaded lipid-polymer hybrid nanoparticles (PE-LPN) were
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successfully prepared and evaluated for physicochemical and anticancer effects. Nanosized PELPN was obtained with a perfect spherical morphology. PE-LPN exhibited the controlled release
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of two drugs in a sequential manner. The nanoparticles showed typical endocytosis-mediated cellular uptake in cancer cells. The ratiometric combination of PTX and ETP was significantly
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more cytotoxic than the individual drugs. Note that a superior cytotoxic effect was observed for
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dual drug–loaded PE-LPN compared to a cocktail combination at a much lower dose. Similarly, PE-LPN showed significantly higher apoptosis of cancer cells (~45%) compared to other groups
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with greater caspase-3 and -8 activity. Note that PE-LPN showed a remarkable tumor regression effect and exhibited 2-fold superior efficacy compared to free drugs. The group treated with PE-
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LPN showed significantly fewer Ki-67-positive cells (less than 25%) than the groups treated
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with PTX/ETP or a single drug, which suggests less active cell proliferation and a considerably
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greater tumor growth inhibition effect. The results show that combining drugs can significantly improve the therapeutic properties of chemotherapeutic drugs. The therapeutic efficacy of ETP and PTX (a powerful anticancer drug) for treating osteosarcoma is improved when the drugs are combined in a polymer-lipid hybrid nanoparticle system.
Acknowledgement This study was supported from the grant of Luoyang Orthopedic Hospital of Henan Province, China.
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Figure captions Figure 1: Schematic illustration of preparation of paclitaxel and etoposide-loaded lipid-polymer
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hybrid nanoparticles Figure 2: (a) Particle size distribution of PE-LPN (b) transmission electron microscope (TEM)
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image of PE-LPN. (c) In vitro drug release profile of PTX and ETP from PE-LPN in PBS buffer.
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The release study was carried out in PBS at 37°C. Results are expressed as mean±S.D (n=3). Figure 3: In vitro cellular uptake of PE-LPN formulations in MG63 cells. CLSM analysis of
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MG63 cells after treatment with PE-LPN after 2h incubation (DAPI dye: blue, and rhodamine-B:
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red).
Figure 4: In vitro cytotoxicity of blank nanoparticles at various concentrations against MG63.
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The cells were incubated with blank NP for 24 h and the cytotoxicity assay was performed by
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MTT reagent. Results are expressed as mean±S.D (n=8).
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Figure 5: In vitro cytotoxicity of free PTX, free ETP, free PTX/ETP, and PE-LPN against MG63. The cytotoxicity assay was performed by MTT assay. **p<0.01 is the statistical difference between PE-LPN and PTX and ETP, respectively. Results are expressed as mean±S.D (n=8). Statistical differences were tested by one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test
Figure 6: Flow cytometer analysis of cell apoptosis using annexinV-FITC and PI staining. The MG63 cells were exposed with free PTX, free ETP, free PTX/ETP, and PE-LPN at a concentration of 1000 ng/ml and incubated for 24 h. ***p<0.001 is the statistical difference between PE-LPN and PTX and ETP, respectively. Results are expressed as mean±S.D (n=3).
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Statistical differences were tested by one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test
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Figure 7: Caspase-3 and 8 activities were measured as a second parameter of apoptotic cell death in a MG63 cancer cells. Significant increase in apoptosis was observed when they were treated
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with combinational nanoparticles. **p<0.01 is the statistical difference between PE-LPN and PTX and ETP, respectively. Results are expressed as mean±S.D (n=3). Statistical differences
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were tested by one-way analysis of variance (ANOVA) followed by the Dunnett post-hoc test
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Figure 8: (a) Antitumor efficacy study in terms of tumor volume in MG63 xenograft tumor
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te
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Results are expressed as mean±S.D (n=6).
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model (b) immunohistochemical analysis of tumor mass for Ki67+ (C) histogram of Ki67+ cells.
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