br different time intervals The drug release experiments for
different time intervals. The drug release experiments for each media were conducted in triplicate.
2.6. Conjugation of HER2 Apt to CCM-loaded HSA NPs
The conjugation of HER2-binding Apt to the surface of HSA NPs was achieved via covalent amide bond formation between amine groups of Apt and carboxyl groups of albumin NPs. Carboxyl groups of HSA were activated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) . Briefly, HSA NPs (5 mg/mL) was suspended in 1 mL PBS buffer (10 mM, A 61603 7.4) contain-ing NHS (0.2 mg/mL, 2 mM) and EDC (1 mg/mL, 5 mM) stirred slowly for 3 min at room temperature . Then, the resulting NHS-activated nanoparticles were collected by centrifugation (16,000 × rcf, 3 min, 4 °C) and redispersed in 200 μL of DNase/RNase free water. The activated nanoparticles were reacted with modified 3′-NH2 and 5′-Cy5 HB5 DNA aptamer with different concentrations (1, 5, 10 and 20 μg, in DNase/ RNase free water) under stirring for 4 h at room temperature. Then, the stirring was allowed to proceed for 12 h at 4 °C. Finally, Apt conju-gated nanoparticles were purified by centrifugation (2 × 5 min, 16,000 ×g, 4 °C) and redispersed in DNase/RNase free water using ultrasonication for 30 s. For clarity, CCM-loaded HSA nanoparticles with and without aptamer were designated as Apt-HSA/CCM NPs and HSA/CCM NPs, respectively. The grafting amount of aptamer was quan-tified using a fluorescence spectrophotometer (Cary Eclipse, Varian) by comparing intensity of the Cy5–HB5–NH2 solution before and after reaction.
2.7. Evaluating the conjugation of HER2 Apt to HSA NPs
To confirm the conjugation of HER2-binding Apt to the surface of NPs, gel retardation assay was performed . To this end, electropho-resis was performed using agarose gel 2% (w/v) containing ethidium bromide (0.5 μg/mL final concentration) in a Tris-acetate-EDTA buffer 1 M. HSA/CCM NPs, purified and non-purified Apt-HSA/CCM NPs were mixed by 2 mL of loading dye buffer and loaded into gel agarose. Free HER2-binding Apt was used as control.
In order to confirm the conjugation of HER2 Apt to HSA NPs, the chemical structure of NPs was characterized by Fourier transform infra-red spectroscopy (FTIR). To this end, 2 mg of each sample was mixed with 100 mg of KBr and made into pellets. The spectra were recorded for over a range of 4000–400 cm−1 using FT-IR spectrophotometer (Perkin Elmer Spectrometer Frontier, America).
2.8. In vitro cellular uptake
The cellular uptake of free CCM, non-targeted and Apt-targeted HSA/ CCM NPs by MCF7 and SK-BR3 cells was investigated by fluorescence microscopy (Zeiss). Briefly, MCF7 and SK-BR3 cells were cultivated at a density of 1 × 104 cells per well in 24-well plates for 24 h at 37 °C. Then, the cell culture medium was removed and the cells were incu-bated by free CCM, HSA/CCM NPs and Apt-HSA/CCM NPs containing equivalent concentration of 5 μM CCM in culture medium (DMEM) for 3 h at 37 °C. Then, the treatment medium was removed and the cells were washed with pre-warmed PBS three times, fixed with 4% parafor-maldehyde for 5 min at room temperature. After fixation, the cells were incubated with DAPI (0.1 μg/mL) for 5 min in order to staining the nu-clei, rewashed two times with PBS and visualized by fluorescent microscopy.
2.9. In vitro cytotoxicity assays
To evaluate and compare the anti-proliferative effect of free CCM, non-targeted and Apt-targeted HSA/CCM NPs; cell viability (MTT) assay was carried out with HER2 positive SK-BR3 and HER2 negative MCF-7 breast cancer cell lines. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin (100 μg mL−1), streptomycin (100 U mL−1), and 10% heat-inactivated FBS. The cells were kept in at 37 °C humidified atmosphere with 5% CO2. For MTT assay, 105 cells were seeded into 96-well cell culture plates and incubated at standard condition to reach exponential growth. Next day, the cells were treated with different concentration (0, 15, 30, 50, and 70 μM) of either free curcumin dissolved in DMSO or equiv-alent doses of curcumin-loaded HSA nanoparticle formulations and in-cubated for 48 h. For the preparation of curcumin solution, curcumin was first dissolved in DMSO as a 10 mM stock solution and finally di-luted in cell culture medium so as to remain the amount of DMSO 0.1% v/v of the culture medium [17,28]. After incubation time, the me-dium was removed and the wells were washed twice with 1× PBS buffer. Then, the cells were incubated with 100 μL of MTT solution (0.5 mg/ mL MTT diluted in PBS) for 4 h at 37 °C and 5% CO2. Subse-quently, the MTT solution was removed and DMSO (100 μL) was added to the wells. Then, the optical density (OD) at 600 nm was re-corded using a plate reader. The percentage of cell viability for each treatment was calculated as the ratio of the mean OD of separate wells (n = 3) to that of control in which DMSO has been added at concentra-tion of 0.1% v/v of cell culture medium [17,28].