Loading of Dox onto Fe2O3-PDA nanoparticles and pH-triggered release
Ultraviolet–visible spectra (Fig. S1a) reveal that Fe2O3-PDA-Dox and Dox have similar characteristic peaks at approximately 478 nm, indicating that the Fe2O3-PDA nanoparticles successfully carried the Dox molecules. The in vitro drug-release curves of Fe2O3-PDA-Dox (Fig. S1b) indicate that in a PBS solution of neutral pH (7.4), the Fe2O3-PDA-Dox nanoparticles released roughly 2.0% of their Dox within 10 h and 7.5% of their Dox within 28 h; at a pH of 6.5, simulating the acidic environment of tumor tissue, 29 and 32% of the Dox was released at 10 h and 28 h, respectively.
Magnetic properties of Fe2O3-PDA-Doxnanoparticles
Concentration-dependent (0–200 μg/mL) signal attenuation (Fig. S2) was seen in T2WI images, indicating the feasibility of using MRI to monitor the in vivo treatment process and verifying the use of Fe2O3-PDA-Dox nanoparticles as a negative MRI contrast agent.
Photothermal response of Fe2O3-PDA-Dox nanoparticles
The nanoparticle temperature increased steadily with increasing in vitro irradiation time (Fig. S3a). The heating curve (Fig. S3b) shows that the temperature gradually increased as the Fe2O3-PDA-Dox concentration increased from 0 to 20 μg/material. After irradiation for 300 s., the temperature of pure water increased only 3.5 °C (room temperature, 24.5 °C) but increased roughly 35 °C at a nanoparticle concentration of 20 μg/mL, which was the greatest temperature increase.
In vivo MRI monitoring of Fe2O3-PDA-Dox distribution
T2 MRI images were acquired at 1 h and 24 h after injection following the injection of Fe2O3-PDA-Dox nanoparticles (after CA4P injection in some groups) via the hepatic artery. The in vivo Fe2O3-PDA-Dox content of the tumor was assessed (Fig. 1). T2 image signals from the tumor were even lower at 24 h than at 1 h in both groups, indicating that most of the Fe2O3-PDA-Dox had entered the tumor tissue. The T2 signal in the CA4P + pTACE group was lower than that of the pTACE group at both 1 h and 24 h after injection, which indicates that the relaxation time of the tumor tissue was shorter in the CA4P + pTACE group, and the tumor tissue in this group contained more Fe2O3-PDA-Dox particles. This observation indicates that CA4P promotes the uptake of Fe2O3-PDA-Dox by tumor tissue.
Neutron activation analysis of tissue Fe content
The mean Fe content of tumor tissue in the CA4P + pTACE group at 1 h and 24 h after injection was greater than that of normal liver tissue (23.72 ± 12.45 μg/g and 14.61 ± 8.23 μg/g; normal tissue, 4.66 ± 1.52 μg/g and 7.67 ± 1.35 μg/g, respectively). The mean Fe content of tumor tissue in the pTACE group differed much less from that of normal tissue at 1 h and 24 h after injection (5.66 ± 4.29 μg/g and 2.76 ± 1.33 μg/g; normal liver, 3.53 ± 1.23 μg/g and 8.58 ± 2.64 μg/g, respectively) (Fig. 2). This result indicates that while there was no significant difference in liver uptake of Fe2O3-PDA-Dox between these two groups, Fe2O3-PDA-Dox uptake was significantly higher in the in tumors treated with CA4P + pTACE than in those treated with pTACE alone.
Photothermal ablation
The temperature of the tumor area rose significantly in both groups following the injection of Fe2O3-PDA-Dox nanoparticles. In the pTACE group, the temperature increased to 42 °C and 55 °C at 1 and 5 min after near infrared laser irradiation, respectively. Greater temperature increases were observed in the CA4P + pTACE group, to 47 °C and 62 °C at 1 and 5 min after irradiation, respectively. The control group displayed a temperature increase of less than 5 °C following irradiation. The resulting heating curve (Fig. 3) indicates that co-injection of CA4P promotes the uptake of Fe2O3-PDA nanoparticles by tumor tissue, with the temperature in the tumor area rising to over 60 °C within 5 min after irradiation. This temperature can effectively kill tumor cells.
MRI assessment of tumor treatment effectiveness
MRI examination of liver tissue in the control, pTACE, and CA4P + pTACE groups was performed before and at 3 d, 7 d, and 10 d after injection to dynamically assess tumor necrosis and changes in tumor volume (Fig. 4). On day 10, the tumor volume was 7.53 ± 1.72 cm3 (control), 2.14 ± 0.24 cm3 (pTACE), and 0.43 ± 0.11 cm3 (CA4P + pTACE). The tumors in the control group grew steadily, and T2WI images displayed a slightly elevated signal. By comparison, the tumor volume was significantly lower in the pTACE and the CA4P + pTACE groups, and T2WI images displayed a slightly reduced signal. While the difference in volume change between the pTACE group and control group at 10 d was not significant, that between the CA4P + pTACE group and controls differed significantly.
Pathology and immunohistochemistry results
H & E staining revealed that rats in the pTACE and CA4P + pTACE groups had small numbers of malformed, disarranged tumor cells, some of which appeared in the form of broad stripes. Necrosis was visible in most tissues, and infiltration by small numbers of inflammatory cells, as well as moderate amounts of fibrous tissue, were observed. Microscopic analysis of TUNEL-stained tissues revealed apoptosis in 26.27 ± 5.65% of the tumor cells in the pTACE group and 31.58 ± 6.82% of the tumor cells in the CA4P + pTACE group. Differences in the expression of tumor markers between the pTACE and CA4P + pTACE groups as indicated by immunohistochemical staining were as follows: Ki67, 0.75 ± 0.67% vs 0.42 ± 0.42%; CD31 (MVD),10.6 ± 4.16% vs 8.40 ± 4.01%; VEGF, 148.63 ± 42.48% vs 129.76 ± 36.03%, respectively. The low level of Ki-67 expression indicates significant tumor cell necrosis, apoptosis, and inhibition of proliferation in both groups after treatment. Angiogenesis was lower in tumors of animals treated with CA4P + pTACE than in those treated with pTACE alone, and the expression of CD31 and VEGF was qualitatively reduced accordingly (Fig. 5).
Safety: Hepatorenal toxicity
Transient liver and kidney toxicity were observed in both groups on day 3. The CA4P + pTACE group sustained more damage than did the pTACE group. The toxic effects gradually decreased on days 7 and 10. By day 10, the AST, ALT, and CREA levels were normal. Indicative of bile duct damage, TBIL levels increased slightly in both groups (Fig. 6), with no significant difference between the groups.