Synergistic delivery of 5-fluorouracil and curcumin using human serum albumin-coated iron oxide nanoparticles by folic acid targeting
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Human serum albumin is the most abundant protein in plasma with the ability to bind to a variety of drug molecules. Magnetic nanoparticles are being extensively used in drug delivery due to its intrinsic magnetic properties. In this work, we have synthesized human serum albumin-coated citrate-functionalized iron oxide nanoparticles by CDI coupling. Furthermore, folic acid was decorated on human serum albumin by EDC and NHS coupling to confer targetability. Two cytotoxic drugs 5-fluorouracil (5FU) and curcumin were co-delivered. Wherein, the former is an anticancer agent and latter is a drug resistance depressor of former. The nanoparticles showed good aqueous dispersibility with a zeta potential of − 49.1 mV and magnetic core size in the range of 10–15 nm, thus exhibiting good magnetic property with magnetic saturation of 33.59 emu/g. Controlled drug release behavior was noticed in both drugs with faster release profile of 5FU. Nanoparticles also showed good cytotoxicity with lower IC50 values in the presence of magnetic field. The contrasting difference was noticed in folic acid-decorated and non-decorated composites, similarly in the presence of magnetic field where cell uptake was enhanced.
KeywordsHuman serum albumin Magnetic nanoparticles 5-Fluorouracil Curcumin Folic acid
Despite the advancement in cancer treatment modalities, such as surgical intervention, radiation, and chemotherapeutic drugs, cancer yet remains one of the world’s most catastrophic diseases; with more than 10 million new cases every year (Hamzehalipour Almaki et al. 2017). Extermination of healthy cells and systemic toxicity lower the quality of patient’s life. Inability to administer therapeutic moieties to acquire selectivity of the desired targets with marginal or no collateral damage has largely accounted for the discrepancy (Peer et al. 2007).
The growth of nanotechnology has unfolded a new paradigm of possibilities in the development of medical sciences like drug delivery field, leading to the development of new drug carrier composites in the nanometer size (Lohcharoenkal et al. 2014; Roco 2011). Nanoparticles, comprising active pharmaceutical ingredients with biocompatible materials in nanometer size, have shown enhanced anticancer property, improved pharmacokinetics, pharmacodynamics, and active intracellular delivery (Lomis et al. 2016). Concurrently, nanoparticles (NPs) are being developed as drug carriers. Thanks to careful nanostructure construction (tailored drug release characteristics, low immunogenicity, etc.), yielding improved treatment efficacy and reduction of unwanted side effects (Veiseh et al. 2010).
Among myriad non-therapeutic constructs for effective and safe therapeutics, protein-based carriers are ideal because of: greater shelf life, amphiphilicity, amenability for surface modification, biodegradability, and ease of ligand bioconjugation (Tarhini et al. 2017). HAS is a major plasma protein involved in the maintenance of osmotic pressure and transport of nutrients to the cells. Its high aqueous solubility (up to 40% w/v) at pH 7.4 and its binding ability to a wide variety of drugs has made it ideal for drug delivery application (Lohcharoenkal et al. 2014). Hence, HAS is used in this work. Many drugs and endogenous molecules are known to bind to albumin. These properties, as well as its preferential uptake in tumor and inflamed tissue, have made it an ideal material for drug delivery (Elzoghby et al. 2012; Kratz 2008).
Magnetic nanoparticles (MNP) like Fe3O4 due to its intrinsic magnetic properties have attracted curiosity that enables magnetic targeting, magnetic resonance imaging, magnetic hyperthermia, etc. In addition to these physicochemical profiles, MNP can be easily tailored for a functional group, inexpensive production, and possesses non-offensive toxicity profile (Lu et al. 2007; Veiseh et al. 2010). Moreover, Fe3O4-based products like Feraheme®, Feridex I.V.®, and Gastromark® have already gained approval for biomedical applications (Revia and Zhang 2016). Upon metabolism, iron ions are added to the body’s iron stores and eventually incorporated by erythrocytes allowing for their safe use in vivo (Sun et al. 2008).
5-Fluorouracil (5FU), an anti-metabolite drug, has been attributed to incorporate fluoronucleotides in place of nucleotides that inhibits the nucleic acid synthetic enzyme thymidylate synthase (TS), hence, exhibiting cytotoxicity. It is being used in the treatment of a range of cancers namely colorectal cancer, breast cancers, and cancers of the aerodigestive tract. However, dihydropyrimidine dehydrogenase (DPD), an enzyme abundantly expressed in liver, catabolizes more than 80% of administered 5FU to dihydrofluorouracil (DHFU) (Longley et al. 2003). Furthermore, 5FU also up-regulates several survival signals including NF-kB and Akt. NF-kB pathway is a major downstream effect or pathway leading to chemoresistance (Vinod et al. 2013).
Curcumin (CUR), a natural polyphenol from the root of Curcuma longa Linn, is a pharmacologically safe. Although hydrophobicis yet potent anticancer molecule against a variety of cancers including breast cancer (Yu et al. 2014; Kakran et al. 2012; Rachmawati et al. 2013). Many mechanisms are involved in the biological activities of CUR including NF-kB, IkBa kinase, Akt, activator protein-1, mitogen-activated protein kinases (MAPK), cyclooxygenase-2 and 5-lipoxygenases, inducible nitric-oxide synthase, urinary plasminogen activator, tumor necrosis factor, chemokines, and cell cycle machinery which have been suggested as the targets of CUR (Choi et al. 2008; Li and Zhang 2014). Curcumin downregulates NF-kB, both directly or via TS pathway, thereby circumventing 5FU resistance. Thus, down regulation of NF-kB by chemopreventives is an effective mechanism to tackle drug resistance (Vinod et al. 2013). However, the limited clinical utility of curcumin is due to its poor solubility and unstable at neutral and basic pH (Saengkrit et al. 2014).
Folic acid (FA), vitamin B9, is vital for the maintenance and proliferation of all the cells. Folic acid receptors are overexpressed on the surface of many human tumor cells, including ovarian, lung, breast, endometrial, renal, and colon cancers (Sun et al. 2006). Significant up-regulation of the folate receptor on tumor tissue has led to the hypothesis that folate-linked therapeutic agents might display reduced off-site toxicity and enhanced potency against tumor cells compared to non-targeted drugs (Xia and Low 2010). FA and FA conjugates can bind to the FRs with high affinity and enter cells by receptor-mediated endocytosis, so the FA-modified drug delivery vectors can transfer the therapeutic agents to tumor cells that exhibit amplified foliate receptor expression. In case of normal cells, FRs’ expression is much lower (Lin et al. 2016).
In this work, we have synthesized folic acid-decorated human serum albumin-coated Fe3O4 nanoparticles (C-MNP-HSA-FA) for synergistic delivery of 5-fluorouracil and curcumin for the treatment of breast cancer. This work attempts to entrap 5FU to circumvent its metabolism and further lower chemoresistance of 5FU using CUR. HSA confers CUR solubility and higher bioavailability.
Materials and methods
Anhydrous iron (III) chloride (FeCl3), iron (II) chloride tetrahydrate (FeCl2·4H2O) 99%, ammonium hydroxide (30–33% M), Tween-80, folic acid (FA), N,N′-carbonyldiimidazole (CDI), (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinamide (NHS) were purchased from Sigma-Aldrich. Oleic acid and sodium citrate were purchased from TCI chemicals. 5-Fluorouracil and curcumin were purchased from MOLchem. Nitrogen-purged Mili-Q was used in all the steps involved in the synthesis and formulation of magnetic nanoparticles.
Synthesis of iron oxide magnetic nanoparticles (MNP)
Iron oxide nanoparticles were prepared by alkaline co-precipitation method (Yallapu et al. 2011; Ahn et al. 2012). Briefly, to the solutions of 0.1 M Fe3+(100 mL) and Fe2+(50 mL) at 80 °C 10 mL of 25%, ammonia solution was added dropwise for 1 min with constant stirring at 6000 rpm. A 50 mL of 0.005 M trisodium citrate was added and stirred for 30 min under an inert atmosphere (N2). Thus, obtained citric acid-coated Fe3O4 (C-MNP) were washed multiple times by magnetic decantation using milliQ water and stored at 4 °C.
Coating of magnetic nanoparticles with HSA by CDI coupling
C-MNP of 100 mg was suspended in MilliQ water by sonication. C-MNP suspensions were washed with water and DMSO ratio (1:4, 2:3, 3:2, and 4:1 v/v) and finally washed three times with anhydrous DMSO to eliminate traces of water. To the anhydrous C-MNP, 1 g of CDI was added. The reaction mixture as was allowed to stir at room temperature for 6 h. The C-MNP suspension was washed with DMSO to obtain CDI-free MNP CDI adducts.
C-MNP CDI adduct was added dropwise to 100 mL of 1 mg/mL of a buffered solution of HSA. The reaction mixture was allowed to stir for 12 h. The unreacted HSA was washed with phosphate buffer saline by centrifugation to obtain serum albumin-coated magnetic nanoparticles (C-MNP-HSA).
FA conjugation to C-MNP-HSA
The FA was coupled covalently on the surface of C-MNP-HSA nanoparticles using zero-length crosslinkers, EDC and NHS (Yang et al. 2014). To 5 mg of folic acid, dissolved in 10 mL of dry DMSO, 15 mg of NHS and 30 mg of EDC were added under vigorous stirring. The reaction mixture was allowed to stir for 1 h in the dark at room temperature to obtain activated FA.
A 50 mg of C-MNP- HSA was dissolved in 100 mL of carbonate buffer solution (pH 9.8, 0.05 M). To this, 5 mL of activated folic acid in DMSO was added dropwise. The reaction was allowed to stir 12 h in the dark at room temperature. Folic acid-decorated nanoparticles were washed multiple times by centrifugation.
Physical and chemical characterization of nanoparticles
Fourier transform infrared spectroscopy (FTIR) of MNP, C-MNP, C-MNP-HSA, and C-MNP-HSA-FA was recorded using Thermo Nicolet 8700, USA in 4000–500 cm−1 range, with KBr as the sample holder.
The hydrodynamic particle-size measurements and surface charge of MNP, C-MNP, C-MNP-HSA, and C-MNP-HSA-FA were measured by dynamic light scattering using Horiba SZ-100 particle-size analyzer. The size measurement was carried out in polystyrene cuvettes and zeta potential was measured using a graphite electrode polystyrene cuvette. All the samples were of concentration 1 mg/10 mL, at 25 °C temperature, and pH 7.4.
Particles were analyzed by Philips/FEI Inc., Briarcliff, Manor, NY, transmission electron microscope (TEM). Sample preparation was carried out by a drop of an aqueous dispersion of C-MNP-HSA-FA nanoparticles on a carbon-coated copper TEM grid and was allowed to air dry.
Thermogravimetric analysis (TGA) of MNP, C-MNP, and C-MNP-HSA-FA was recorded using SDT Q600, USA. A 5 mg sample was placed in the aluminum sample holder and heated at a ramp rate of 10 °C/min to 600 °C under nitrogen environment.
Magnetic properties of MNP, C-MNP, and C-MNP-HSA-FA were measured using Lakeshore, Model 7407, USA, vibrating sample magnetometer (VSM) at 15 kOe applied magnetic field at room temperature.
Drug entrapment efficiency and drug release study
In drug-loading procedure, 5 mg of 5FU and 5 mg of CUR were dissolved in 5 mL of DMSO and were added dropwise to 50 mg aqueous dispersion of C-MNP-HSA-FA. The mixture was allowed to stir for 6 h at room temperature. 5FU and CUR-loaded C-MNP-HSA-FA (5FU-CUR-C-MNP-HSA-FA) was separated by centrifugation.
The drug release was performed by suspending 10 mg of 5FU-CUR-MNP-HSA-FA NPs in 50 mL of phosphate buffer solution (pH 7.4) with 0.05% (w/v) of Tween®80 at 37 °C. At specific time intervals, 1 mL of release buffer was aspirated and replaced by fresh phosphate buffer solution. The concentration of 5FU and CUR was determined using a Hitachi U-2800 UV–Vis spectrophotometer at a wavelength of 241 nm and 421 nm, respectively.
In vitro cytotoxicity assay of drug-loaded nanocarriers
The cytotoxic properties of 5FU, CUR, C-MNP-HSA-FA, 5FU-CUR-C-MNP- HSA, 5FU-CUR-C-MNP-HSA-FA, and 5FU-CUR-C-MNP-HSA-FA under magnetic field (5FU-CUR-C-MNP- HSA-FA-Mag) was determined by MTT cell viability assay. MCF-7 cells were seeded onto 96-well plates at 5000 cells per well. After 24 h, the cells were treated with different concentrations (0.01, 0.1, 1, 10, and 100 μg/mL) of 5FU, CUR, C-MNP- HSA -FA, 5FU-CUR-C-MNP-HSA, 5FU-CUR-C-MNP-HSA-FA, and 5FU-CUR–MNP-HSA-FA-Mag. For 5FU-CUR-MNP-HSA-FA-Mag, cells were cultivated under 0.05 T magnet placed beneath the wells. After the incubation, the cells were washed multiple times with DPBS and replaced with fresh growth medium. To assess the cell viability, 50 µL dye solution was added to 100 µL of the medium in each well. The formazan crystals thus formed after incubation for 4 h at 37 °C in 5% CO2 atmosphere was solubilized in 100 µL of DMSO, and the plates were gently shaken to solubilize the formed formazan. The absorbance was then measured with a spectrophotometer at 540 nm.
Quantitative uptake of C-MNP-HAS and C-MNP-HSA-FA in the presence and absence of magnetic field was evaluated by measuring iron content in the cell by atomic absorption spectroscopy (AAS) (Prijic et al. 2010; Dinda et al. 2012). MCF-7 were seeded in 6-well plates, upon reaching 90% confluence, the supernatant was discarded and replaced by 2 mL of nanocomposite suspension in culture medium (iron content of 200 µg/mL). Plates were then placed in a 37 °C, 5% CO2 incubator for the duration of 60 and 240 min. After incubation, cells were trypsinized and washed with DPBS three times to ensure the complete removal of media and leftover nanoparticles. The cell pellets of each well were then digested by adding 1 mL of HCl. After 24 h, dilution at 1:4 was made and the iron content of the cell lysate was analyzed by AAS.
Drug release study, MTT assay, and cellular uptake data were collected in triplicates and analyzed statistically using SPSS 20 for standard mean and standard deviation.
Results and discussion
Fourier transform infrared spectra
Size and zeta potential
Size and zeta potential analysis of MNP, C-MNP, C-MNP-HAS, and C-MNP-HAS-FA
Mean particle size (nm)
Vibrating sample magnetometry
Entrapment efficiency and in vitro release of drugs
In vitro cytotoxicity of drug-loaded NPs
The results conclude that C-MNP-HSA-FA shows good aqueous suspending ability, and can be prepared and loaded with 5FU and CUR. Nanoparticles showed superparamagnetic behavior thus suitable for drug delivery applications. The controlled release of both the drugs was elicited by 5FU-CUR-C-MNP-HSA-FA. Drug-loaded nanoparticles showed a dose-dependent cytotoxic effect in MCF-7 cells. Enhanced cytotoxicity and cell uptake were observed in case of folic acid-conjugated nanoparticles, due to folic acid targeting. The external magnetic field can further enhance cytotoxicity and cellular uptake of nanoparticles due to magnetic targeting effect. Moreover, nanoparticles showed good magnetic saturation value and, hence, can be used to enhance treatment efficacy by magnetic targeting.
Compliance with ethical standards
Conflict of interest
Authors Chinmay G Hiremath, Mahadevappa Y Kariduraganavar, and Murigendra B Hiremath do not have a conflict of interest for publishing an original article entitled “Synergistic delivery of 5-Fluorouracil and Curcumin using Human Serum Albumin-coated Iron Oxide Nanoparticles by Folic Acid targeting” in your journal.
This article does not contain any studies with human participants or animals performed by any authors.
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