br Statistical analysis br Statistical
2.10. Statistical analysis
Statistical significance was determined with the unpaired Student's t-test for two-group comparison and two-way ANOVA for multiple-group analysis performed with GraphPad Prism 6.00; differences be-tween groups have been considered significant at a p value of b0.05.
3. Results and discussion
3.1. Preparation and characterization of HSA/CCM nanoparticles
In this study, with the aim of improving the potency of anticancer properties of CCM to treat breast cancer cells, human serum albumin was used as a drug carrier for encapsulation of this MitoPY1 drug molecule. Previously, water-soluble CCM-loaded human serum albumin nanoparticles were prepared using albumin bound technology . In the present work, desolvation technology according to what reported before by Langer et al.  with minor modification was used for the preparation of CCM-loaded HSA NPs.
Fig. 1 shows SEM image of HSA NPs prepared in this work along with their size distribution histograms. The obtained HSA/CCM NPs had a spherical morphology and smooth surface (Fig. 1A). To determine the size distribution histograms, dimensions of several NPs were measured from their SEM images using ImageJ 1.42 . From this histogram (Fig. 1B) the mean diameter of NPs and its standard deviation was de-termined to be dc = 183 ± 46.3 nm.
The hydrodynamic diameter and zeta potential of NPs were ana-lyzed by DLS (Table 1). The value of zeta potential of HSA/CCM NPs was −25 ± 2.7 mV. This negative charge is due to the presence of car-boxylic groups in HSA molecules. The hydrodynamic diameter was 246.1 ± 15.4 nm, significantly larger than the diameter in SEM image due to the presence of bulk water molecules surrounding NPs in aqua medium.
The obtained HSA/CCM NPs by desolvation method demonstrated the yield of 82.8%, drug loading efficiency of 3.4%, encapsulation effi-ciency of 71.3% and good stability. Also, the solubility of curcumin in aqueous solution has increased from 0.0004 mg/mL for free curcumin
Fig. 1. SEM image of HSA/CCM NPs dried on an Aluminum grid. The scale bar corresponds to 500 nm. (B) Size distribution histogram, plotted as number of NPs N (dc) that have a diameter of dc.
to 0.16 mg/mL in the form of curcumin-loaded human serum albumin nanoparticles.
The in vitro release behavior of curcumin from HSA/CCM nanoparti-cles was investigated in four different release buffer conditions includ-ing PBS (pH 7.4) and acetate buffer (pH 5.5) with and without GSH (10 mM), at 37 °C during 48 h; the results of which are presented in Fig. 2 as cumulative percentage release.
In the case of PBS media at pH 7.4, the amount of CCM released was near zero during the first 3 h, and it reached to 6% ultimately over 48 h which is attributed to the stability of nanoparticles under physiological conditions. As expected, the slow release of CCM from HSA NPs in PBS indicates that little drug release exists during blood circulation and min-imizes its side effects.
Two modified media of PBS and acetate buffer (pH 5.5) containing 10 mM GSH were applied for stimulating the reductive environment of cytosol and lysosomes, respectively. For PBS containing 10 mM GSH, about 32% of the total loaded CCM was released in the first 6 h. After the burst release, a constant slow release up to 40% of the loaded CCM was observed within 48 h, showing a prolonged drug release. For acetate buffer (pH 5.5) containing 10 mM GSH, in comparison with PBS media containing 10 mM GSH, the initial release was about 48% in the first 6 h and eventually reached to 58% after 72 h (Fig. 2) which has increased slightly. This release behavior is beneficial for the con-trolled release of drugs at tumor sites and inside the cells, because of the lower pH value and reductive environment of tumor and endosomal compartments compared to serum environment.
In this study, the release of CCM from HSA NPs prepared via desolvation method was assessed in GSH containing media. As shown in Fig. 2, the presence of GSH accelerates the release of CCM. Accord-ingly, albumin could be considered as a bioreducible polymer having 17 disulphide bonds which undergo cleavage under reductive environ-ment through thiol-disulphide exchange reaction .
The hydrodynamic diameter, PDI and zeta potential of different nanoparticle formulations (mean ± S.D., n = 3).
Nanoparticles Size (nm) PDI Zeta potential (mV)
3.3. Conjugation of HER2 Apt to the surface of nanoparticles
In order to generate a targeted curcumin-loaded HSA NP formula-tion against HER2 positive cells, HB5 aptamer with high selectivity and binding affinity to HER2 overexpressing cells was selected through bibliographic study [10,31]. The modified Cy5-HB5-NH2 aptamer was used to accomplish aptamer-conjugated HSA NP (apt-HSA NP) using EDC/NHS reaction. To confirm the conjugation, FTIR spectra of free Aptamer, HSA nanoparticle, and Aptamer conjugated HSA nanoparticles are presented in Fig. 3A. In the spectrum of Apt, peak assignments are in accordance with the literature . The characteristic bands relevant for DNA/RNA are in the spectral range 1800–800 cm−1. As shown in Fig. 3A, conjugation of Aptamer DNA molecules onto HSA NPs confirms by the appearance of the characteristic peaks between 900 and 1200 cm−1 in the spectrum of Apt-HSA NPs, which could be assigned to the sugar phosphate vibration. By comparing the spectrum of Apt-HSA NPs with HSA NPs and free Apt spectra, it can be seen that the distinct peaks at 925, 1044 and 1110 cm−1 are appeared in both of Apt and Apt-HSA NPs spectra while those are absent in HSA NPs spectrum.