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Drug loading refers to a relative amount of drug bound to the nanoparticles. Drugs are bound to the nanoparticles mainly by chemical bond. However, some limitations of drug-loaded nanoparticle such as low drug loading, fast drug release existed.[3,4] Dopamine is a small molecule mimic of the adhesive component (L-DOPA) of marine mussels with the chemical structure of catecholamine. Dopamine aqueous solutions can be used as adhesives since dopamine adsorbs on almost all kinds of surfaces, in particular on metal surfaces by forming strong coordination bonds.[5,6]
Hydrophobic anti-cancer drug, doxorubicin (DOX),[7,8] has shown remarkable antitumor efficacy in patients afflicted by mammary gland tumor. However, the major drawback of DOX remains its low aggregation in target tumor sites and high toxicity to non-target organs, thus limiting its therapeutic usage in curing the patients of breast cancer.[8,9] Therefore, there is an urgent clinical need to find applicable therapeutic strategies that especially target the drug to tumor sites thus enhancing the tumor cells killing and lessening total drug exposure during treatment.
Recentlyï¼Œtargeted drug delivery system including aptamers peptides  and antibodies[11,12], has already become increasingly prevalent in cancer therapy. Through localizing drugs, it can both improve drug efficacy and reduce the adverse effects of drugs on normal tissues. However, targeted drug delivery in vivo has not fulfilled its expectations, because most targeted drug delivery systems can penetrate onlya fewcell diametersinto the extravasculartumortissue.[14,15] Small molecules (carbohydrates, folate, and cholesterol), peptides, growth factors, and aptamers could be used as targeting ligands for specific drug release[16-18]. Folic acid play a vital role in the development and progression of breast cancer. Studies have found that folic acid receptor expressed abundantly in solid tumors just like advanced mammary carcinoma. Thus, folic acid could be used as a targeting agent.
Here, a simple and effective approach was developed to fabricate nanoparticle-based delivery platform, which could provide promising drug loading, pH-responsive drug release, and tumor-killing capability. The resulted IO nanoparticles are composed of Fe3O4 core and Folic shell. Additionally, the copolymer comprises two unique dopamine surfaces, which could be taken as adhesives. The anti-cancer agent, DOX, is embedded in the copolymer’s matrix, tightly attaching to the nanoparticles via the adhesion of dopamine. Meanwhile, the PEG moiety in the copolymers helped the nanoparticle well dispersible in aqueous solution. These structures are illustrated in Fig 1.
Fig. 1 Schematic of Fe3O4 nanoparticle drug delivery
The MDA-MB-231 cell, human breast cancer cell line, was purchased from American Type Culture Collection (ATCC). Roswell Park Memorial Institute (RPMI) 1640, fetal bovinserum (FBS) and Trypsin were purchased from Life Technologies (Gibco). Cell culture incubator was purchased from Heraeus (Germany). CCK-8 Assay Kit was from manufacturer Dojindo Laboratory (Japan).
Synthesis and characterization
Dynamic light scattering (DLS)
Particle size distribution (mean diameter and polydispersity index), and zeta potential were captured by a particle analyzer Malvern Zetasizer (Malvern Instruments, UK).Prior to measurement, samples were suspended in distilled water (pH6.5 and PH7.4)and then measured at a temperature of 25â„ƒ and a scattering angle of 90 degree. The mean diameter±standard deviation were calculated applying multimodal analysis. Values reported are the mean value±standard deviation for two replicate samples.
Human breast cancer cells (MDA-MB-231), were grown in RPMI 1640 with 10% fetal bovine serum, 100U/ml penicillin, and 100μg/ml streptomycin. Cellswere placed in an incubator at 37â„ƒ with a humid atmosphere of 5% CO2 and the culture medium was refreshed every day.
Immunofluorescence staining assays
Cellular uptake and intracellular distribution of nanoparticles was tracked by ordinary fluorescence microscopy and confocal laser scanning microscopy by incubating the MDA-MB-231 cells with free DOXã€DOX-loaded Folic/Dopamine nanoparticles or DOX-loaded Dopamine nanoparticles for 30 min, 1 h, 2 h, 24 h, 48 h, 72 h. MDA-MB-231 cells were seeded in the culture dish with a cover slip at a density of 2×105 cells/dish for 24 h. Then the cells were added with free DOX, DOX-loaded Folic/Dopamine nanoparticles or DOX-loaded Dopamine nanoparticles. After a predetermined incubation time, the cover slip was washed with cold PBS for three times. The cells were fixed by 4% paraformaldehyde at room temperature for 15 min, followed by incubation with DAPI for 30 min. Then the cover slip was set on a microscope slide and examined by fluorescence microscopy and CLSM using a blue laser405nmfor excitation and free DOX, DOX-loaded Folic/Dopamine nanoparticles or DOX-loaded Dopamine nanoparticles detected using red laser568 nm respectively.
The cytotoxicity of free DOX, DOX-loaded Folic/Dopamine nanoparticles and DOX-loaded Dopamine nanoparticles was performed using a Cell Counting Kit-8 assay (CCK-8). The MDA-MB-231 cells were seeded at a density of 2000 cell per well in 96-well culture plates and cultured at 37â„ƒ in 5% CO2. After 24 h cultivation, the medium was replaced with fresh (RPMI) 1640 solution containing free DOX, DOX-loaded Folic/Dopamine nanoparticles and DOX-loaded Dopamine nanoparticles ( the DOX concentration ranged from 1 to 100 mg/mL, six well per sample). After being incubated for 24h, 48h, 72h, 10 μL CCK-8 solution and 90 μL medium was added to each well and incubated for another 2 h at 37 â„ƒ. Thereafter, the absorbance of each well was measured using a microplate reader at the wavelength of 450 nm.
Results and discussion
Preparation and characterization of Folic/Dopamine nanoparticle
Fig.2 (A) Diameter and size distribution of DOX-loaded Folic/Dopamine nanoparticles (B) TEM images of blank nanoparticles (C) TEM images of DOX-loaded Folic/Dopamine nanoparticles.(bar: 100nm)
The hydrodynamic size of the Folic/Dopamine/DOX nanoparticle measured by DLS was 203.2 nm (Fig.2A) and no marked changes in size were observed for three times of experiments. TEM images showed that the Folic/Dopamine/DOX nanoparticle remained well dispersed and free of aggregation in water solution (Fig.2C) compared to blan nanoparticle (Fig.2B).
Fig.3 The size change of DOX-loaded Folic/Dopamine nanoparticles in response to PH decreasing from 7.4(A) to 6.5(B) determined by DLS measurement. The size change of DOX-loaded Dopamine nanoparticles in response to PH decreasing from 7.4(C) to 6.5(D) determined by DLS measurement.
To gain a better understanding of the pH sensitivity of the nanoparticles, the pH-induced size changes zeta-potential of the developed nanoparticles were observed by DLS (Fig.3). After adjusting the pH value of the Folic/Dopamine/DOX nanoparticle solution to 7.4, the average size was about 203.2 nm (Fig.3A); when the pH value changed from 7.4 to 6.5 (Fig.3B), there was a sharp decrease of the average diameter of the particle (437.8 nm) (Fig.3B). Concerning the Dopamine/DOX nanoparticles (blank Folic),the nanoparticles showed larger size (399.8 nm) at pH 6.5 (Fig.3D) compared to those prepared at pH 7.4 (169.5 nm) (Fig.3C), indicating that DOX might release from the nanoparticles in response to acidic environment. Additionally, after adjusting the pH value to 6.5, aggregation of nanoparticles was observed, with diameter ranging from (122.4 nm) to (396.1nm) in Folic/Dopamine-modified nanoparticles, and from (122.4) to (615.1) in Dopamine-modified nanoparticles.
Fig.4 Release and distribution of doxorubicin visualized by confocal microscopy. Time course of uptake and release of doxorubicin for the DOX-loaded Folic/Dopamine nanoparticles (A) and Free-DOX (B). Nuclei were stained with DAPI (blue), doxorubicin is red.
The cellular uptake and intracellular distribution of free-DOX and DOX-loaded Folic/Dopamine nanoparticles were investigated using fluorescence microscope and CLSM in MDA-MB 231 cell line. As shown in (Fig.4), after the first 30 min incubation with the free-DOX in MDA-MB 231 cells, DOX was visibly observed in cell cytoplasm and nuclei. The distribution of DOX in the cytoplasm and nuclei was rarely observed in the case of DOX-loaded Folic/Dopamine nanoparticles. While after 2 h incubation with DOX-loaded nanoparticles, the DOX was found to be aggregated in and around the cytoplasm. After treatment for 24 h, there was significantly increased amount of DOX distributed around the nuclei, while the intensity of DOX fluorescence observed in nuclei was a little weaker than that of free DOX. Through observing the morphology of the cells, it was not hard to find out that, after 48 h incubation, the cell skeleton was shrunk and the cells looked unhealthier in comparison to the shorter time incubation samples, which may be caused by the DOX-loaded nanoparticles’ successful delivery of the drug into the nuclei and the later actions of DOX[12, 30].
Fig.5 The cell inhibition rate of Free-DOX, DOX-loaded Folic/Dopamine nanoparticles and DOX-loaded Dopamine nanoparticles as the function of DOX concentration.
The cytotoxicity to MDA-MB 231 cells of the DOX-loaded nanoparticles was determined using the cck-8 assay. As shown in (Fig.5), the toxicity of DOX enhanced with the increase of its dose. After incubation for different concentration of free DOX and DOX-loaded Folic/Dopamine nanoparticles, the latter exhibited lower toxicity compared to the free DOX. Additionally, the cell-killing effect of DOX-loaded Folic/Dopamine nanoparticles was concentration and time dependent over the range 1-100 µg/mL. In higher concentration, the DOX-loaded nanoparticles exhibited obviously faster increase in cell inhibition efficiency than free DOX. As the time went on, the DOX-loaded nanoparticles exhibited relatively same role in cell inhibition efficiency with free DOX in higher concentration. The possible reason is that small molecules like doxorubicin can have an easier access to cells rapidly, whereas the macromolecules such as drug-loaded nanoparticles have to be endocytosed to enter the cells.
According to this study, DOX-loaded Folic/Dopamine nanoparticles were synthesized. The anti-tumor drug doxorubicin, targeting molecule Folic acid and adhesives dopamine aqueous solutions were immobilized. The Folic/Dopamine/DOX nanoparticles exhibitted low toxicity to normal cells, while promising cellular uptaking to kill breast cancer MDA-MB-231 cells. Above all, the DOX-loaded Folic/Dopamine nanoparticles might exert an excellent potency as a carrier platform for anti-tumor drug delivery.