br To assess the conjugation efficiency different amount
To assess the conjugation efficiency, different amount of modified Cy5-HB5-NH2 aptamer was used for bioconjugation and the grafting amount of aptamer was quantified by fluorescence spectroscopy by comparing the intensity of Cy5-HB5-NH2 aptamer solution with the su-pernatant of purified NPs. The results showed that the conjugation effi-ciency of Apt was 1 μg Apt per 1 mg NPs.
Fig. 2. In vitro drug release from HSA/CCM nanoparticles in different AzBTS-(NH4)2 at 37 °C.
Fig. 3. (A) FTIR spectra of Apt, Apt conjugated HSA NPs, and unconjugated HSA NPs. (B) Agarose gel electrophoresis. (a) DNA ladder, (b) Unconjugated HSA/CCM NPs, (c) free Apt,
(d) supernatant of Apt-conjugated HSA/CCM NPs after purification, (e) Apt-conjugated HSA/CCM NPs after purification and washing (f) supernatant of Apt-conjugated HSA NPs after purification (g) Apt-conjugated HSA NPs after purification and washing.
The attachment of Apt to the surface of NPs was investigated by aga-rose gel electrophoresis and the results are shown in Fig. 3B. Unconju-gated HSA NPs did not show any band (Fig. 3Bb) while free Apt showed a significant band on the gel (Fig. 3Bc). After purification, a light band corresponding to the existence of the residual unconjugated Apt was visible in the supernatant (Fig. 3Bd and f). In the case of purified Apt-HSA NPs and Apt-HSA/CCM NPs, the observed band in loading site confirms the successful performance of conjugation and shows that bounded-Apt cannot migrate under the applied electrophoresis condi-tion (Fig. 3Be and g).
Characterization of the Apt conjugated nanoparticles is presented in Table 1. The functionalization of the NPs with Apt resulted in a 35 nm increase in particle size (from 246.1 ± 15.4 to 281.1 ± 11.1 nm) and a slight decrease in zeta potential (from −25.1 ± 2.1 to −33.3 ± 2.5 mV), presumably attributed to the presence of Apt on the NPs sur-face. No significant change in drug loading efficiency occurred after conjugation.
3.4. Cellular uptake study
The aim of the present study is to investigate the targeted delivery of HSA/CCM NPs to HER2 positive breast cancer cells so that the therapeu-tic effects of the associated drugs could be improved. To this end, the in vitro cell culture of SK-BR3 (a HER2 positive breast cancer cell) and MCF-7 (a HER2 negative breast cancer cell) in the presence of HSA/ CCM NPs and Apt-HSA/CCM NPs was performed to assess the targeting properties of these NPs. Taking the advantage of green intrinsic
fluorescence of curcumin, uptake of free curcumin and curcumin-loaded HSA NPs by SK-BR3 and MCF-7 cells can be appropriately moni-tored under a fluorescence microscope.
The obtained images indicate that both of the cells treated with free curcumin and HSA/CCM NPs had insignificant intracellular fluorescence, which are not comparable to cells treated with Apt-HSA/CCM NPs (Fig. 4). As it is demonstrated, the green fluorescence intensity was much higher in the cells treated with HER2 Apt conjugated nanoparti-cles (Apt-HSA/CCM NPs) compared to HSA/CCM NPs treated cells. The higher fluorescence intensity in the cells treated with Apt-HSA/CCM was attributed to the enhanced cellular uptake through HER2 receptor. Moreover, it is obviously demonstrated that green fluorescence inten-sity in SK-BR3 cells treated with Apt-HSA/CCM NPs is significantly higher than MCF-7 cells due to overexpression of HER2 in SK-BR3 cells enables more cellular internalization, suggesting that Apt-decorated nanoparticles have an excellent targeting ability to HER2-positive tumor cells. In another study Wang et al.  reported that HB5-conjugated mesoporous silica–carbon nanoparticles had significantly increased accumulation within HER2-overexpressed SKBR-3 cells com-pared to unmodified ones. This group demonstrated that the intracellu-lar uptake of targeted nanoparticles was performed through receptor-mediated endocytosis .
As it is demonstrated in Fig. 4, the uptake of the nanoparticles by cells was significantly enhanced when the nanoparticles were deco-rated with anti-HER 2 Apt in comparison with the non-targeted ones supporting again that the presence of Apt facilitates uptake of nanopar-ticles by the cells.
Fig. 4. Fluorescent microscopy image of free curcumin, Apt-targeted and non-targeted HSA/CCM NPs in (HER2+) SK-BR3 and (HER2−) MCF-7 cell lines.
For both of the cell lines, no significant difference in the fluorescence intensity was observed between free CCM and non-targeted HSA/CCM NPs treated cells during incubation time.
3.5. Anticancer activity of curcumin-loaded nanoparticles
The in vitro cytotoxic effect of free CCM, CCM-loaded HSA NPs and aptamer-conjugated CCM-loaded HSA NPs was investigated on HER2 positive (SK-BR3) and HER2 negative (MCF-7) cell lines after 24, 48 and 72 h incubation. MTT assay was performed using equivalent dos-ages of free CCM and CCM-loaded HSA nanoparticles in both of targeted and non-targeted formulations. Toxicity of the empty HSA nanoparticles and Apt-HSA nanoparticles was also evaluated in parallel. Our control experiment showed no significant toxicity associated with either bare HSA nanoparticles or Apt-HSA nanoparticles without the drug even at concentrations higher than its highest corresponding amount in the CCM loaded nanoparticle formulation used for drug cytotoxic study. It indicated that Apt-HSA NPs could be useful as drug carriers due to high biocompatibility.