Hydrodynamic diameter size distribution and
Hydrodynamic diameter, size distribution and zeta potential of nanoparticles were determined via dynamic light scattering (DLS) (Brookhaven Instrument, USA).
Morphology of the nanoparticles was identified by scanning electron microscopy (SEM). The SEM procedure was performed by drying of NPs suspension, gold sputter coating, and examining with SEM (MIRA\ \TESCAN) using acceleration voltage of 10 kV.
2.4. Drug loading and encapsulation efficiency
In order to determine drug loading efficiency (DLE) and drug encap-sulation efficiency (DEE), CCM-loaded HSA nanoparticles were isolated by centrifugation (20,000 ×g, 20 min at 4 °C). The supernatant was col-lected and amount of free CCM was measured via spectrophotometer at 420 nm. The amount of entrapped CCM in NPs formulation was calcu-lated by subtracting the amount of free CCM from the total CCM. Drug loading efficiency (DLE) (%) and drug encapsulation efficiency (DEE)
DLE ð%Þ ¼ Weight of entrapped CCM in NPs 100 ð1Þ
The total weight of nanoparticles
DEE ð%Þ ¼ Total Weight of initial CCM used–free non−entrapped CCM Total Weight of initial CCM used
2.5. In vitro drug release study
Drug release from NPs was studied at 37 °C in four different condi-tions, as described previously with minor modifications , including:
10 mM GSH which resembles the intracellular condition, (3) acetate buffer pH 5.5 similar to microenvironment of tumor and (4) acetate buffer pH 5.5 containing 10 mM GSH corresponds to the lysosomal en-vironment . 0.5% SDS was used in all release Pronase E to maintain a sink condition and facilitate the release of CCM in buffer media . Then, accurately weighed of CCM/HSA NPs solution was divided in a number of Eppendorf tubes (1 mg/mL). The tubes were incubated at 37 °C and shaken at 150 rpm. At predetermined time intervals, each tube was centrifuged at 3000 rpm for 30s to separate the released (pelleted) CCM from the nanoparticles. 1 mL of ethanol was added to pellet CCM due to insolubility of free CCM in water. Therefore, CCM was re-dissolved in solution and the absorbance was measured spectro-photometrically at 428 nm to determine the amount of CCM released in
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, pH 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.