br was observed between HUVEC
was observed between HUVEC Talaporfin sodium (ME2906) incubated with free DOX and [email protected] NPs. However, MDA-MB-231 cells exposed to [email protected] NPs exhibited significantly higher mean fluorescence in-tensity than those treated with DOX, confirming the NPs are taken up to a greater extent by the cells. This can be explained by a combination of three factors: (1) the EPR effect making it easier for the NPs to enter MDA-MB-231 cells than HUVECs; (2) the active targeting effect of the FA moiety of [email protected]; (3) cellular accumulation of DOX is increased with the NPs because the presence of OA inhibits MRP mediated efflux. The cellular uptake observed here with the [email protected] NPs is notably higher than for other FA functionalized CS systems reported in the literature [40,41], indicating the significant chemosensitizing effect of OA.
3.5. Pharmacokinetics and biocompatibility
The pharmacokinetics of free DOX and [email protected] NPs were studied in SD rats. As can be seen in Fig. 5A, DOX concentrations in the plasma exhibited bi-exponential declines over 24 h. The mean plasma concentrations of the [email protected] NPs were considerably higher
than those of the free DOX. Moreover, the AUC, terminal t1/2, and MRT values of the [email protected] NPs are significantly increased (P < 0.05) in comparison to the DOX group. This may arise because of reduced distribution of the [email protected] NPs from the central compartment into the tissues and organs. This is thought to be related to the physicochemical properties of NPs (i.e., diameter, zeta potential, and architecture) [34,42]. The prolonged blood circulation of [email protected] NPs should lead to improved antitumor efficacy.
To explore the biocompatibility of the NPs, a hemolysis assay was conducted to evaluate their compatibility with the blood. The amount of hemoglobin released into solution is directly proportional to the hemolytic activity of the NPs. Almost no visible hemolysis can be ob-served for the FA-CS-g-OA NPs at concentrations below 250 μg/mL (Fig. 5B). Free DOX induced RBC lysis even at very low concentrations, whereas 250 μg/mL [email protected] NPs had much lower hemolytic activity (< 10%). Although free OA causes some damage to RBCs, when OA was grafted onto the FA-CS backbone it showed dramatically lower levels of hemolysis. This suggested that FA-CS-g-OA NPs are blood-compatible materials as well as potent drug delivery systems.
Fig. 4. Cellular uptake assays performed with MDA-MB-231 and HUVEC cells. (A) CLSM data depicting cellular uptake of DOX loaded NPs by MDA-MB-231 and HUVEC cells (all scale bars = 50 μm) and (B) flow cytometry analysis.
3.6. In vivo and ex vivo fluorescence imaging
The real-time biodistribution behavior of free DiR and DiR loaded NPs ([email protected]) was monitored using the live imaging system after intravenous injection into xenografted MDA-MB-231-tumor-bearing mice. A visible signal for DiR was detected in the tumor foci at 4 h after administration of [email protected] NPs (Fig. 6A), the in-tensity of which increased up to 8 h and was maintained up to 24 h. In comparison, free DiR is distributed much more widely throughout the animal; the presence of DiR in the tumor was barely noticeable at 12 h or earlier, and only after 24 h is a dim fluorescence visible in the tumor area. These findings are probably due to rapid blood clearance within the tumor. To observe the distribution of DiR more clearly, mice were sacri-ficed 24 h post-injection, and their hearts, livers, spleens, lungs, kidneys and tumors were harvested for ex vivo imaging (Fig. 6B). While DiR in the [email protected] NPs is clearly concentrated in the tumor, with some in the liver, when free DiR is injected it is distributed throughout the body, with noticeable presence in the heat, lungs, liver, spleen and kidneys as well as in the tumor. Quantitative region of interest (ROI) analysis (Fig. 6C) reveals that the tumors of mice injected with [email protected] NPs exhibited a much higher fluorescence efficiency (2.5-fold greater) than free DiR. These results all show that [email protected] NPs can effectively target and release payloads selectively to a human MDA-MB-231 tumor in mice, due to the combined influence of the EPR effect and FA receptor mediated cell uptake.