FT-IR spectrum. ( a ) sebacic acid ( b ) polysebacic anhydride. 

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... plotted versus time. All experiments were carried out in triplicates. The anti-tumor activity of the prepared pH responsive NCs was examined on the MDA-MB-231 breast cancer cells purchased from the American Type Culture Collection (ATCC). Cell viability assay included blank wells containing medium only, untreated control cells and cells treated with the empty NCs, free DOX and DOX-loaded nanoparticles. Briefly, the breast cancer cells were seeded in 96-well plates, incubated for 24 h and treated by 100 μ L sterile PBS (blank groups) as well as PBS solution containing unloaded nanocapsules (blank NCs), free DOX (0.6 μ g/mL) and DOX containing nanocapsules (PEG-PLH-PSA-DOX-NCs) (0.6 μ g/mL and 1.2 μ g/mL). After 3 days of incubation at 37°C and 5% CO 2 , cell viability was estimated by adding 10 μ L of WST Kit-8 that was added to each well and incubated for 2 h. The absorbance values of the wells were obtained by UV spectroscopy (Wallac Victor plate reader, Turku, Finland) at 450 nm. To visualize the intracellular uptake of DOX containing nanocapsules by the cells, confocal laser scanning microscopy was employed. To accomplish this, MDA-MB-231 cells (15,000 cells per well) were seeded in 8-well glass slide (Lab- Tek II, NY, USA), incubated for 24 h and then treated with sterile PBS (0.01 M), PBS containing free DOX (0.6 μ g/mL) and nanocapsules containing the same amount of DOX (PEG-PLH-PSA-DOX-NCs). After 3-days incubation, the medium was removed and the cells were rinsed twice with sterile PBS (0.067 M, pH 7.4). Fixing was accomplished by adding 400 μ L of cold paraformaldehyde (PFA, 4%) and by incubating for 15 min at room temperature. Excess paraformaldehyde was removed by rinsing with sterile PBS (0.067 M, pH 7.4). After fixing, the cell nucleus was labeled with DAPI (4 ′ ,6-diamidino-2-phenylindole) by adding one droplet of pro- long gold antifade reagent containing DAPI (Invitrogen, Oregon-USA) to each well. Fixed cells were covered by cover slips, kept at 2 – 8°C for 24 h and they were inspected by a Zeiss LSM 710 confocal microscope, equipped with Plan- Apochromatic 63×1.4 NA oil immersion objective (Zeiss- Germany). The image processing and visualization were performed by using the ZEN 2011 software (Carl Zeiss, Germany). Cell uptake of nanocapsules by cancerous cells was also examined by fluorescence microscopy. In this experiment, MDA-MB-231 breast cancer cells were seeded in 8-well glass slide (15,000 cells per well) and treated with sterile PBS as a control group, free DOX and Coumarin-labeled PEG-PLH- PSA-DOX-NCs. Treated cells were incubated for 1 day and fixed by PFA (4%) as described above. The influence of PEGylation of nanocapsules on macrophage uptake was analyzed on human acute monocytic leukemia cells (THP-1) that were obtained as a kind gift from Dr. Lina Prasmickaite and grown in RPMI 1640 cell culture medium (Lonza, Verviers, Belgium) containing 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM Glutamine. THP-1 cells were seeded (15,000 cells per well) in 8-well glass slide and induced to differentiate into macrophages by adding 200 μ L culture medium containing 10 μ M TPA (12-O- tetradecanoyl-phorbol-13-acetate) and incubated at 37°C in the presence of 5% CO 2 . After 48 h, the macrophages were treated by replacing TPA containing medium with sterile PBS, PEGylated NCs (PEG-PLH-PSA-DOX-NCs) and non-PEGylated NCs (PLH-PSA-DOX-NCs) with DOX concentration of 0.6 μ g/mL. Treated macrophages were incubated for 6 h and fixed by paraformaldehyde (4%) as described above for the microscopy study. From three represen- tative experiments, we analysed 10 cells with the uptake of non-PEGylated beads and 17 cells with the uptake of PEGylated beads. The level of total Doxorubicin fluorescence in the individual cells was measured as a function of relative difference in integrated intensity to the nucleus (DAPI) label- ing of the cells. No saturated pixels were present and no background subtraction was performed. The individual nucleus was manually masked by freehand selection to avoid any discrepancies due to size, fluorescence intensity or shape. The total fluorescence intensity of the DOX loaded into PEGylated and non-PEGylated particles was measured with ImageJ [17]. Data is presented as mean and standard deviation, and the statistical significance was tested with a two- tailed Mann – Whitney test. The values having p ≤ 0.0014 was considered significant (GraphPad, Prism). The H-NMR spectra of both sebacic acid and polysebacic anhydride are compared in Fig. 2. It was found that the peak related to the OH groups located at 10.49 ppm, representing the monomer structure [3, 14] has disappeared in the spectrum of the synthesized polysebacic anhydride. This phenomenon is related to the conversion of carboxylic acid groups to anhydride. In addition, FTIR analysis confirmed the synthesis of the polysebacic anhydride. The FTIR spectra of both sebacic acid and polysebacic anhydride are shown in Fig. 3. The spectrum of sebacic acid showed characteristic absorption bands at 1,697, 1,300 and 930 cm − 1 , representing carboxylic acid groups (Fig. 3a). The broad band presented at 3,335 – 2,500 cm − 1 was ascribed to the strong hydrogen bond- ing of the -OH groups of the free acid. In the case of the spectrum for the polymer (Fig. 3b), these bands disappeared and the polysebacic anhydride characteristic absorption band was observed at 1,816 – 1,740 cm − 1 . A sharp peak in the range of 1,090 – 1,030 illustrates stretching of the anhydride groups ( − CO-O-CO-) [12]. Peaks observed in the domain of 2,920 and 2,870 cm − 1 are related to stretching of C-H bonds in CH 2 groups. Results obtained from GPC demonstrated that the molecular weight of the prepared PSA is about 2,500 Da with a polydispersity index (PDI) of 1.30. Although the main strategy of this study was to target DOX via pH-responsive nanocapsules, the enhanced permeability and retention (EPR) effects of nano-size particles were also expected to contribute to the entrapping of nanocapsules to the solid tumor [18]. To exploit the EPR effect in tumor targeting, relatively small nanocapsules are required [3]. The results from dynamic light scattering (DLS) for DOX loaded NCs, before coating with PLH and PEG (PSA-DOX-NCs), are shown in Fig. 4. The size distribution of particles was rather broad, but most of the particles were located in the range of 150 – 350 nm. SEM micrographs exhibited a fine spherical morphology for PSA-DOX-NCs (Fig. 5). The drug delivery systems are usually defined as a delivery technique to carry at least two-fold dosage of pharmaceutical agents compared to conventional drug administration [19]. Hence, encapsulation efficiency and loading capacity of NPs are important for sustained delivery. The challenge for polymer-based drug delivery systems is to obtain a sufficiently high drug loading capacity. The water-soluble DOX exhibits weak amphipathic properties (pK a 8.3) and it is generally difficult to encapsulate the drug from an aqueous medium. There are also various challenges related to improving the DOX loading efficiency by preventing its migration from the organic to the aqueous phase, which is the main cause of the high amount of non-loaded drug in the supernatant. In some studies, cooperation of DOX with anionic polymers [20, 21] and interaction of an anionic surfactant [22] was used to develop DOX encapsulation efficiency up to 42.5 and 49.3%, respectively. In our study, encapsulation efficiency and loading capacity of DOX in polysebacic anhydride based nanocapsules were estimated to be 48 and 5.3%, respectively. These values are close to the reported [20 – 22] values for encapsulation of DOX in nanocapsules. In the literature there are plenty of studies aimed to improve encapsulation efficiency of highly hydrophilic DOX. It was shown that encapsulation efficiency of DOX can be significantly improved by either using negatively charged polymers [23] or co-encapsulation of DOX with other gradients such as ammonium sulfate [24]. Consequently, NCs fabricated in this study via the double emulsion method exhibited acceptable drug encapsulation for DOX delivery, without any modification of the polymer to entrap more DOX. Poly (L-histidine) (PLH) is a biodegradable polyamino acid. The polymer backbone has many imidazole functional groups with pKa around 6.0 and PLH exhibits buffering properties in the physiological pH range. Imidazole groups can be protonated at pH below 5.8, and as a consequence PLH can be dissolved in dilute acids [25]. This feature has been used to design different PLH based complexes as pH-sensitive delivery systems. For instance, PLH has been applied either as an outer shell or in cooperation with alginate to prepare microcapsules used for protein delivery [26]. In these studies, PLH was defined as a promising compound for protein delivery. In order to test the pH-response of poly (L-histidine), DOX loaded NCs (PSA-DOX-NCs) were coated with PLH by adsorbing PLH molecules on the NCs surfaces through elec- trostatic interactions. For this purpose, zeta potential of both DOX loaded NCs and PLH were measured in dilute acid medium with the intension to create the same condition needed for coating. The results showed zeta potential values of +29.4 and − 35 mV for PLH and PSA-DOX-NCs, respectively. The results favor the conjecture that positively charged PLH can be adsorbed onto negatively charged NCs. To provide more evidence for this hypothesis, PLH-coated NCs (PLH- PSA-DOX-NCs) were characterized by FTIR to establish whether the absorbance peaks coming from PLH functional groups can be detected in the FTIR spectrum of the NCs. Figure 6 shows FTIR spectra for DOX loaded nanocapsules (PSA-DOX-NCs) (Fig. 6a), DOX loaded nanocapsules coated with PLH (PLH-PSA-DOX-NCs) (Fig. 6b), and pure PLH used as blank (Fig. 6c) to compare the success of the coating. A comparison of FTIR-spectra reveals that the spectrum ...