Archive | 2019
Curcumin nanocrystallites are an ideal nanoplatform for cancer chemotherapy
Abstract
Curcumin, a principal active component extracted from rhi-zomes of turmeric, has a promising clinical activity against a wide variety of tumors. It’s extremely low solubility in water is the great limitation in the application of cancer chemotherapy. Nanocrystals have recently emerged as a promising pharmaceutical preparations way for the poorly water soluble drugs. Accordingly, we prepared curcumin nanocrystals (CNs) with a mean diameter of 15 nm by quick emulsion freeze-drying method. CNs proved to be effective in drug delivery, such as avoiding uptake by the reticuloendothelial system (RES), prolonging the circulation times of curcumin as well as enhancing the well-known permeation and retention (EPR) effect, as a result of which CNs accumulate at the tumor site. Furthermore, we design to conjugate a targeting ligand-cyclic iRGD peptide to CNs in order to selectively deliver Curcumin to cancer cells with greater efficiency and enhance the permeability of cells leading more drugs inside cells. Hence, our results suggested that iRGD-CN, which be of both characteristics of passive and active targeting, may be a potential drug delivery system in enhance the effective therapy of curcumin chemotherapy. *Correspondence to: Li W, Department of Neurosurgery, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518039, China, [email protected]” Li M, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China Received: September 06, 2019; Accepted: October 11, 2019; Published: October 15, 2019 Introduction In the preparation of CNs, Pluronic®F-127 were used as emulsion stabilizer and eventually a small amount of them wrapped in the surface of the CNs [1,2]. Pluronic®F-127 are nonionic triblock copolymers composed of a central hydrophobic chain of poly (propylene oxide) (PPO) flanked by two hydrophilic chains of poly (ethylene oxide) (PEO)(PEO-PPO-PEO) [3,4]. These amphiphilic triblock copolymers strengthen stealth properties to CNs, allowing them to reduce potential toxicity and avoid uptake by the RES, which is crucial for achieving long circulation time in blood and enhanced uptake by tumors via EPR effect [5-7]. In addition, the thin coating of Pluronic®F-127 also provide a binding site for cyclic iRGD peptide, which is active targeting ligand enhancing the therapeutic efficacy. iRGD (CRGDK/RGPD/EC) is a kind of tumor-penetrating peptide which can contribute CNs to increasing cancer vascular and tissue permeability in a αv integrin and neuropilin1(expressing at the surface of tumor blood vessel endothelial cells and tumor cells)–dependent manner. iRGD-based targeting mechanism for tumor is that the iRGD motif mediates binding to integrins on tumor endothelium and a proteolytic cleavage then exposes a binding motif for neuropilin-1, which mediates penetration into tumor tissue and cells. Compared with traditional targeting moiety simply paying attention to the ability to identify tumor cells and lacking capacity of leading drugs penetration into tumor tissue and cells, iRGD peptide overcomes this limitation and establishes a capability for tissue/cells-penetrating drug delivery [1,8]. Current research shows that the microenvironment in solid tumors is very distinct from that in normal tissues. Due to deregulated cancer cell metabolism, highly heterogeneous vasculature and defective blood perfusion, the tumor microenvironment is characterized high interstitial pressure and cell density [9,10]. How to make CNs penetrate tumor blood vessel endothelial cells and tumor tissue is the key to eliminate cancer cells, especially to extirpate interior cancer stem cells [11,12]. Thus, we design an efficient DDS through employing the CNs to be chemically conjugated to the peptide. Materials and methods Materials Pluronic®F-127, 4-(dimethylamino)pyridine (DMAP), triethylamine, and succinic anhydride were purchased from Sigmae-Aldrich (St. Louis, MO, USA). Uppsala 87 Malignant Glioma (U87) were supplied by American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium (DMEM), RMPI 1640 medium, fetal bovine serum (FBS) were provided from Gibco BRL (Grand Island, NY, USA). Curcumin, Cremophor EL, Cell Counting Kit-8 (CCK-8), Rhodamine-phalloidin, and dimethylsulfoxide (DMSO) were purchased from Sigmae-Aldrich (St. Louis, MO, USA). Oregon Green® 488 Taxol, Flutax-2, DAPI (4,6-diamidino-2-phenylindole), Hoechst, LIVE/DEAD Viability/ Cytotoxicity Kit, and Cy5 was bought from Molecular Probes (Eugene, OR, USA). Taxol (TA) was supplied by Merck (Germany). DSPEPEG-iRGD were supplied by GL Biochem Ltd. (Shanghai, China). Anhydrous ethanol and dichloromethane were purchased from Beijing Chemical Reagent Company (Beijing, China). Pluronic®F-127 functional group modification Terminal hydroxyl groups on Pluronic®F-127 were converted to carboxyl groups in order to efficiently coupling of iRGD peptide to the CNs surface. The modification of Pluronic®F-127 functional group involves the following procedure. Pluronic®F-127 (0.2 g) was dissolved Wang X (2019) Curcumin nanocrystallites are an ideal nanoplatform for cancer chemotherapy Front Nanosci Nanotech, 2019 doi: 10.15761/FNN.1000186 Volume 5: 2-4 in THF (10 mL), and then DMAP (12 mg), triethylamine (100 μL) and succinic anhydride (600 mg) were added. The reaction mixture was stirred for 48h at room temperature. The solution was dried by a rotary evaporator and was then dissolved in of chloroform (75 mL). The excess succinic anhydride was removed by filtration with a 0.45 μm PTFE membrane syringe filter. The Pluronic®F-127-COOH (F127) was purified by precipitation with ice-cold diethylether [13,14]. The product was identified by 1H-NMR spectrometer (Bruker AVANCE III 600 MHz) and Fourier Transform Infrared Spectrophotometer (JASCO FT/IR-660). Preparation and characterization of CNs and iRGD-CNs CNs were prepared via oil-in-water emulsions freeze-dried method, which generally comprised three steps process. The first step is emulsion preparation. The oil phase (O) was dichloromethane solution containing 40 mg/mL curcumin. Containing 1 wt% Pluronic®F-127COOH aqueous solution was the water phase (W). The volume ratio of oil phase and water phase was fixed at 1:20, and the mixture was mixed through ultrasonic (100 w, 10 min) emulsification to obtain emulsion (O/W). Secondly, the above emulsion was further added into 20-fold the volume of the water phase, and the mixture was frozen by liquid nitrogen followed by freeze-drying using a vacuum freeze-dryer. Finally, CNs suspension was obtained when lyophilized products were dispersed into the water and free surfactant were removed by ultracentrifugation (800000 rcf, 10min). In this study, CNs were functionalized modifications targeted by DSPE-PEG-iRGD ligand were embedded to the surface of CNs during the process of oil-in-water emulsions freeze-dried. In addition, we used fluorescent dye Cy5 instead of curcumin in the preparation progress to obtain nanocrystals with fluorescence properties so as to study the cellular uptake of CNs and the target of iRGD. Characterization of CNs Emulsion droplet size and the desired degree of droplet size uniformity play a key role in the preparation of CNs. Hence, we measured the average particle size and size polydispersity index (PDI) of droplets using Zetasizer (Nanoseries, Malvern, U.K.). CNs size and morphology were determined by transmission electron microscope (TEM, JEOL, Japan) with an acceleration voltage of 200 Kv. The homogeneity of the particles was studied by atomic force microscope (AFM, Bioscope Catalyst). Due to the obvious difference zeta potential of the product during each step of the reaction, the reaction process can be easily detected according to the potential variation. To assess the encapsulation efficiency of curcumin, free curcumin in the CNs suspension was removed by the method of ultrafiltration, and then curcumin containing in CNs was quantified by HPLC. In vitro cellular uptake U87 cells were routinely cultured respectively in complete DMEM and RMPI 1640 medium with 10% FBS, 1% penicillin and 1 % streptomycin in a humidified incubator with 5% CO2 at 37 °C. For the cellular uptake study, U87 cells were seeded at the density of 1 ×105 cells/well in two 24-well cell plates and incubated at 37 °C for 24 h to allow cell attachment. After 24 h, the cells were then incubated with CNs and iRGD-CNs (100 μg/mL) at 37 °C for different time intervals. The cells were rinsed with PBS (pH 7.2) solution softly, pre detached by 0.25% trypsin. The trypsin was removed by centrifugation at 1000 rpm for 5min. The cells were then harvested, re-suspended in 500 ml PBS and examined by flow cytometry using a CyAn ADP 9 color flow cytometer (Beckman Coulter). Using the same method, U87 cells were grown in a culture dish at a density of 1 × 106 cells/dish and incubated at 37 °C for 24h. Following incubation of cells in dishes culture containing 200 μL of CNs and iRGD-CNs. The medium was then removed, and cells were washed with PBS (pH 7.2). Finally, the total fluorescence images of nanocrystals internalized into cells were scanned using an in vivo imaging system (FX Pro, Kodak) with an excitation bandpass filter at 480 nm and an emission at 633 nm. In the confocal laser scanning microscopy (CLSM) imaging studies, U87 cells were first seeded on Petri dishes for 24h, and the medium was replaced with new medium containing 100 μg/mL of CNs and iRGDCNs. There are some differences in the following process of the two cell lines. After incubation for 24 h at 37 °C, the medium of U87 cells was then removed and cells were washed with PBS (pH 7.2) followed by fixing with 4% paraformaldehyde for 30 min. The cell membrane and nuclei were stained with Rhodamine-phalloidin and DAPI, respectively. CNs and iRGD-CNs were detected fluorescence at 633 nm, and the corresponding fluorescent images of the cell membrane and nuclei at 488 nm and 405 nm by CLSM (TCS S