br mV after FA modification
+15.4 ± 0.3 mV after FA modification (because some of the amine groups are converted to amides, and there are COOH groups on FA), and then to −10.3 ± 0.2 mV after subsequent OA grafting. The hy-drodynamic sizes of all the NPs are below 200 nm and they have a narrow size distribution (PDI < 0.45), which is appropriate for tumor targeting by the EPR effect. This size distribution is also suitable for intravenous administration. The [email protected] NPs further ex-hibited a constant size upon storage as an aqueous suspension for 7 days, indicating a good stability to aggregation (Supplementary
The extracellular fluid in the tumor tissue (pH = 6.5–7.2) is more acidic than that in normal tissue (pH∼7.4), while intracellular endo-somes and lysosomes (pH = 4.5–6.5) also possess acidic environments . Therefore, the drug release properties of [email protected] were determined at different pH values to mimic relevant in vitro environ-ments: 1) physiological tissue (pH = 7.4); 2) the extracellular fluid of a tumor (pH = 6.5); and, 3) the endosome-lysosome in carcinoma Calcipotriol (pH = 5.0). The results for DOX release are presented in Fig. 2A. It is clear that pH has a major effect on drug release from the [email protected] DOX NPs. DOX was quickly released at pH 5.0, reaching ca. 75% in the first 24 h, after which it slows. In contrast, only ∼35% of the DOX loading was released after 48 h at pH 7.4. The pH 6.5 experiment is intermediate between these extremes. These effects can be attributed to the increased ionization, and thus solubility, of CS at reduced pHs.
The release profiles of OA from [email protected] NPs follow a very similar trend (Fig. 2B). The cumulative release percentage of OA after 72 h reached 31.0 ± 3.2%, 51.8 ± 5.3% and 76.0 ± 8.2% at pH 7.4, pH 6.5 and pH 5.0 respectively. An acidic milieu can accelerate the release of OA by promoting the hydrolysis of the ester bond connecting OA to CS , in addition to the increased solubility of CS at lower pH. The accelerated release of both active ingredients at weakly acidic pHs is very promising for anti-cancer applications.
3.3. In vitro cytotoxicity and antitumor effects
The in vitro anticancer effects of free DOX, free OA, FA-CS-g-OA and [email protected] NPs were evaluated in MDA-MB-231 human breast cancer cells. Healthy HUVEC cells were used to assess the biocompat-ibility of the various materials. As shown in Fig. 3A, FA-CS-g-OA in-duced negligible cytotoxicity to normal HUVEC cells at concentrations up to 10 µg/mL (> 90% cell viability). OA is slightly toxic to HUVECs, and induces more cell death than FA-CS-g-OA. DOX and [email protected] DOX do cause the death of HUVEC cells. The latter is less toxic than the former, but this effect is not statistically significant (P > 0.05 when the concentration of DOX was less than 10 µg/mL). Nevertheless, this ob-servation is promising for selective delivery applications.
Fig. 2. In vitro release profiles of (A) DOX and (B) OA from [email protected] at different pHs. Error bars represent the standard deviation (n = 3).
All the DOX-containing samples displayed dose-dependent cyto-toxicity to MDA-MB-231 cells (Fig. 3B). Relative to free DOX, [email protected] NPs exhibited greater toxicity to MDA-MB-231 cells over the entire concentration range explored (0.001–10 μg/mL) after 24 h of incubation. Similarly, the cytotoxicity of FA-CS-g-OA was higher than free OA. These effects can be explained by a combination of more ef-ficient cellular uptake in the case of the NPs, and the acidic micro-environment of the cellular milieu accelerating drug release from the formulations.
The half maximal inhibitory concentrations (IC50) of free DOX, free OA, FA-CS-g-OA and [email protected] NPs to MDA-MB-231 cells were calculated to be 7.8 ± 0.9 μg/mL, 123.6 ± 11.7 μg/mL, 15.8 ± 1.7 μg/mL and 1.8 ± 0.1 μg/mL, respectively (Fig. 3C). A sy-nergistic enhancement in cytotoxicity to MDA-MB-231 cells is thus obtained through the integration of two therapeutic agents (DOX and OA) within a single platform. These data further confirm the chemo-sensitizing effect of OA on DOX cytotoxicity as reported in the literature .