br EPR mediated passive targeting and ligands mediated activ
(EPR)-mediated passive targeting and ligands-mediated active tumor targeting are the main benefits of nanoparticles based drug delivery systems [9,10]. Among numerous drug delivery systems, lipid carriers have concerned rising attention due to their high drug-loading capacity, easy preparation, low toxicity and physical stability [11,12]. In addi-tion, nano-carriers are able to co-delivery of anti-cancer agents which has been considered as one of the most promising methods to repel multi-drug resistance (MDR) activity . Co-delivery not only delays the cancer adaptation process, but also reduces drug side effects by decreasing drug doses and achieving synergistic therapeutic efficacy [14,15]. Recently, targeted nanoparticles indicating growing interest regarding their ability to increase therapeutic effectiveness of chemical compounds and reducing possible side effects . Nanostructured lipid carriers (NLC) are classified as Lipid-based colloidal drug delivery systems which are used for efficient co-delivery of antitumor agents [17,18] beside active delivery of drugs through coated ligands for precise conveyance to cancer specific receptors . One of these li-gands is Arginyl-glycyl-aspartic 946414-94-4 (RGD) which is the most common peptide motif that is responsible for cell adhesion to the integrins (especially αvβ3 integrin) in extracellular matrix (ECM) [12,20]. Due to the overexpression of integrins on various cancer cell, it is prospected that RGD-containing nanoparticles will penetrate into the cells more efficiently by integrin-mediated endocytosis [21,22]. The aim of the present study was to prepare and optimize RGD-containing NLC (RGD-NLC) co-loaded with DOX and SC to increase SC’s bioavailability and explore its ability in enhancing the cytotoxic and apoptotic effects of doxorubicin (DOX) on human lung cancer cell line. Furthermore, to investigate molecular mechanism by which SC causes overcoming to DOX resistance, expression of MDR related genes were studied by Real time Polymerase chain reaction (RT-PCR).
2. Material and methods
DOX, 3- (4, 5- Dimethylthiazol- 2- yl)-2, 5- diphenyltetrazolium bromide (MTT), 4′, 6-diamidino-2-phenylindole (DAPI), penicillin-streptomycin, propidium iodide (PI), RPMI 1640 and poloxamer 407 were purchased from Sigma-Aldrich Company (Steinheim, Germany). Precirol® ATO5 (Glyceryl palmito stearate) was prepared from Gattefosse (Saint PeriestCedex, France). Fetal bovine serum (FBS) and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (poly-ethylene glycol)-2000] (DSPE-PEG-2000-amine) were purchased from Gibco (Carlsbad, CA, USA) and Avanti Polar Lipids (Alabaster, AL) re-spectively. A549 cancer cell line was supplied from Pasteur Institute Cell Culture Collection (Tehran, Iran). SYBR green PCR Master Mix and cDNA synthesis kit were obtained from Takara Company (Japan).
2.2. Preparation of RGD-containing NLC co-loaded with DOX and SC
Primarily, DSPE-PEG-amine (2000) and RGD were stirred for 24 h in Dimethyl sulfoxide (DMSO) and the solution was transferred to 1000 Mw cutoff dialysis bag (Sigma-Aldrich, USA) to purify the synthesized polymer. Finally, the modified polymer was obtained after freeze-drying and RGD-PEG-DSPE (yield 73.1%) was achieved. Next, hot homogenization technique used to prepare RGD-containing NLCs for-mulation. For this purpose, Precirol melted as a solid lipid and let to SC and DOX to dissolve or disperse in Miglyol as a liquid oil. Subsequently, RGD-PEG-DSPE was dissolved in aqueous phase containing Poloxamer 407 and added in drop wise manner to the lipid phase under imposed homogenization with high pressure at 20,000 rpm for 30 min at 70 °C (Heidolph, Germany). The temperature of hot oil/water formulation was decreased into ambient or lower temperature in order to re-crys-tallization of NLC-RGD formulation. Process Biochemistry 84 (2019) 172–179
2.3. Characterization of prepared nanoparticles
2.3.1. Particle size, size distribution and zeta potential
The mean diameter, polydispersity index (PDI) and zeta potential of prepared nanoparticles (NPs) were measured by photon correlation spectroscopy (PCS) (Zetasizer ZS, Malvern, UK) and also zeta potential values were determined based on laser Doppler anemometry with the same machine. The samples were put into capillary cells and a minimum of three measurements were provided per sample for zeta potential measurements.
2.3.2. Morphology of nanoparticles
To study the morphology of the particles, the prepared NLCs were diluted with distilled water and visualized by scanning electron mi-croscope (SEM) (KYKY-EM3200, Bio-equip, China) at 26 KV excitation voltage. To provide the gold coated samples, they were placed in dro-plet-by-droplet-manner on glass lamella and then coated under vacuum by a sputter (SC7620-CF, Quorum Technologies, UK).
2.3.3. Drug encapsulation efficiency (EE) and physical stability studies To examine the entrapment efficiency of DOX and SC, the amount of