Characterization of SN38 Loaded uIONPs
The commercial amphiphilic polymer coated uIONPs with an averaged diameter of 3.5 nm was used for encapsulating SN38. The uIONP demonstrated a narrow distribution of hydrodynamic diameter measured by dynamic light scattering (Fig. 1a), indicating monodispersed uIONPs with a highly uniformed size, which was further supported by the transmission electron microscopic (TEM) image (Fig. 1a, inset). After mixing SN38 with uIONPs to allow the accumulation of SN38 in the hydrophobic moiety of the coating polymer, PEG550 was added to further stabilize the loading of SN38. The emerging peak at ~ 380 nm for uIONP/SN38 in the UV-Vis spectrum, comparing to that of uIONP alone and free SN38 peaked at ~ 405 nm(Fig. 1b), indicated the loading of SN38 to the amphiphilic polymer coated uIONPs. Worth noting, the peak of SN38 blue shifted after encapsulation, possibly due to the ionic hydrogen bonding between the Fe and SN38 molecules.
The loading efficiency of SN38 was measured to be 68.2 ± 3.5% and found stable in PBS (pH 7.4), with a <7% variation over 24 hours (Fig. 1c), suggesting the capability of uIONP/SN38 in preventing the premature drug release during blood circulation. At pH of 6.5 and 5.5, which mimicked the acidic conditions in tumor interstitium and lysosome, 47.8% (pH 6.5) and 9.7% (pH 5.5) of SN38 remained encapsulated over 24 hours (Fig. 1c), indicating the potential environment-triggered drug release in the tumor interstitial space and the lysosomes of cancer cells after IGF1-uIONP/SN38 was internalized through receptor-mediated endocytosis. The release profiles of uIONP/SN38 showed a plateau-like stages from one to two hours after exposing to the acidic solutions, indicating a stepwise SN38 release. As the addition of PEG550 further increased the SN38 loading efficiency comparing to that without PEG550 (48.3 ± 6.1%), which showed no plateau-like stage in the release profile (Supporting Information, Fig. s1), the release pattern in Fig. 1c can be ascribed to the fast release of PEG550 with the loosely encapsulated SN38 molecules within one hour, and a subsequent release of SN38 from the inner layer of coating polymer.
Optimization and Functionalization of uIONP/SN38
To investigate the effect of PEG molecules in stabilizing the SN38 loading, PEG with different molecular weights were tested in SN38 encapsulation. The loading efficiencies of SN38 decreased from 68.2 ± 3.5% to 42.1 ± 3.8%, as the molecular weights of PEG increased from 550 to 5000 g/mol (Fig. 2a), indicating a better SN38 loading by the PEG with a lower molecular weight. With PEG550 selected for optimal loading of SN38, the encapsulation of SN38 did not result in a significant (p > 0.05) change on the hydrodynamic diameter (Fig. 2b) or the zeta-potential (Fig. 2c). The uIONP/SN38 was then functionalized with IGF-1 as the targeting ligand for IGF1R, whose expression was highly upregulated in both pancreatic cancer cells and stromal component. The significantly (p < 0.001) increased hydrodynamic diameters of uIONP/SN38 (9.9 ± 1.8 nm) to IGF-uIONP/SN38 (14.5 ± 2.1 nm) indicated the conjugation of IGF-1 (Fig. 2b), which was further confirmed by the change of zeta-potentials from the positive surface charge for uIONP/SN38 (0.99 ± 0.31 mV) to the negative charge (-0.88 ± 0.46 mV) after conjugation of IGF-1 (Fig. 2c).
Targeting of Pancreatic Cancer Cells by IGF1-uIONP/SN38
To validate the targeting effect of IGF1-uIONP/SN38 to pancreatic cancer cells, near infrared (NIR) dye NIR830 labeled IGF1-uIONP/SN38 was incubated with MiaPaCa-2 human pancreatic cancer cells with overexpression of IGF1R[28, 29], with HEK293 human embryonic kidney cells with normal expression of IGF1R as the control cell line. NIR830-IGF1-uIONP/SN38 at the Fe concentration of 0.02 mg/mL was applied to demonstrate the difference in cellular uptake of NIR830-IGF1-uIONP/SN38 by MiaPaCa-2 and HEK293 cells. Extensive NIR signal was observed on MiaPaCa-2 cells (Fig. 3a to c), indicating a substantial uptake of NIR830-IGF1-uIONP/SN38 by the cancer cells. In contrast, NIR830-IGF1-uIONP/SN38 showed undetectable NIR signal on HEK293 cells (Fig. 3d to f). The IGF1-mediated pancreatic cancer cell targeting was further validated by incubating MiaPaCa-2 cells with non-targeted uIONP/SN38, in which no MiaPaCa-2 cell targeting was observed for NIR830-uIONP/SN38 when IGF-1 was not conjugated as the targeting ligand (Fig. 3g to i).
Cytotoxicity of IGF1-uIONP/SN38
The cytotoxicity of IGF1-uIONP/SN38 for targeted pancreatic cancer cells was investigated by measuring the viability of MiaPaCa-2 cells after incubating with IGF1-uIONP/SN38 for 72 hours at 37 °C with the SN38 concentration ranging from 0 to 2500 nM. IGF1-uIONP/SN38 demonstrated a greater inhibitory effect on the growth of MiaPaCa-2 cell than those treated with free SN38 and non-targeted treatment uIONP/SN38, which showed little toxicity (Fig. 4a). The absolute half-maximal inhibitory concentration (IC50) of IGF1-uIONP/SN38 for MiaPaCa-2 cells was calculated to be 11.8 ± 2.3 nM (Fig. 4a). In comparison, MiaPaCa-2 treated with free SN38 reached a viability of ~ 50% at 78.1 nM with no further inhibition on cell growth at higher SN38 concentration (Fig. 4a), suggesting the limit of cytotoxicity induced by free SN38 under the experimental condition. As ~20% cells remained viable after treating with IGF1-uIONP/SN38 at the SN38 concentration of 39.1 to 2500 nM, it can be concluded that IGF1-uIONP as a drug carrier improved the delivery efficiency of SN38 to the pancreatic cancer cells than the free drug, and subsequently enhanced the efficacy of SN38 in inhibiting the cell growth. Meanwhile, IGF1-uIONP/SN38 did not exhibit obvious cytotoxicity for HEK293 cells at most SN38 concentrations, with ~10% reduction of cell viability at the concentration of 2500 nM (Fig. 4b), suggesting the biosafety of IGF1-uIONP/SN38 for non-targeted cells in healthy organs.
Enhancement of Pancreatic Cancer Cell Apoptosis by IGF1-uIONP/SN38
To quantitatively assess the SN38 induced cell apoptosis after the enhanced cancer cell delivery by IGF1-uIONP, IGF1-uIONP/SN38 treated MiaPaCa-2 cells were analyzed by fluorescence assisted cell sorting (FACS) after the fluorescence labeling using an Annexin V Apoptosis kit. After incubating with IGF1-uIONP/SN38 for 24 hours with the SN38 concentrations of 1, 5 and 10 µM, the MiaPaCa-2 cells did not show a dose-dependent change on the populations of dead (AnnexinV+/7-AAD+), apoptotic (AnnexinV+/7-AAD-) and live (AnnexinV-/7-AAD-) cells, with the population of dead cells varying from 80.2 ± 0.9% to 82.2 ± 1.5% (Fig. 5a to d). Meanwhile, MiaPaCa-2 cells treated with free SN38 under the same condition exhibited increased populations of dead cells from 41.8 ± 1.2% to 53.0 ± 1.4% and decreased live cell population from 31.7 ± 1.0% to 23.9 ± 1.2%, when SN38 concentration was increased from 1 to 10 µM (Fig. 5a, e to g). These results suggested that the dosage for IGF1-uIONP/SN38 to be effective in inducing cell apoptosis was remarkably lower than that for free SN38, and IGF1-uIONP/SN38 possessed an approximate 1-fold better efficacy than free SN38 under the same experimental condition. Worth noting, although non-targeted uIONP/SN38 did not exhibited obvious cytotoxicity with the live cell population ranging from 72.5 ± 2.2% to 87.1 ± 1.3% (Fig. 5a, h to j), which was in accordance with the results in cell targeting experiment and cytotoxicity measurement, the dose-dependent decrease of live cell population suggested a minimal apoptotic effect on the cells under current condition, providing a reference for the dosing for future in vivo study.