Synthesis and Functionalization of QD@HMSN NPs
The QD@HMSN NPs were synthesized by a two-step method, as shown in Fig. 1. The QD cores were first coated with dSiO2 through an oil-in-water reverse micro-emulsion silica coating approach [27]. This step effectively integrated hydrophobic QD cores into the silica nanosystem. By modifying the thickness of the dSiO2, we can easily control the size of the hollow cavity after etching. In the second step, as-prepared QD@dSiO2 NPs were further coated with MSN shell and then selectively etched out with Na2CO3 to generate an inner cavity. The final size of QD@HMSN was ~ 72 nm, which has a large cavity (~ 25 nm) and a QD core (~ 5 nm) inside each cavity, showing a distinctly different morphology compared to the QD@dSi2@MSN before etching (Fig. 2a). The surfactant CTAC was later removed via an extraction process by stirring the nanoparticles in NaCl: methanol solution (1 wt%) [28]. The silica pores (~ 2–3 nm) were exposed after the CTAC removal [29], which provide sufficient porous channels for efficient drug loading and release.
Amine groups (NH2) were introduced onto the surface of QD@HMSN by reacting with APTES in absolute ethanol for further functionalization [11]. PEG chains were conjugated onto amine-modified QD@HMSN to improve the solubility in physiological solutions and biocompatibility in vitro and in vivo. NOTA was employed as the coordination chelator for radiolabeling of 64Cu, an excellent radioisotope for PET with the half-life of 12.7 h. TRC105, a chimeric antibody that specifically binds to CD105, was conjugated for efficiently and specifically targeting CD105, which is exclusively expressed on the proliferating tumor vasculature. The hydrodynamic diameter of QD@HMSN was 76.3 ± 8.9 nm based on dynamic light scattering, whereas that of the final conjugate NOTA-QD@HMSN-PEG-TRC105 was increased to 98.1 ± 15.9 nm, suggesting successful conjugation of NOTA, PEG and TRC105 onto the surface of QD@HMSN.
In Vitro CD105 Targeting
Fluorescent-dye FITC was conjugated onto QD@HMSN-PEG-TRC105 for in vitro angiogenesis targeting. As evidenced by flow cytometry results (Fig. 2b), significant enhancement was observed with the targeted group (FITC-QD@HMSN-PEG-TRC105), in comparison with the negative control (PBS), non-targeted group (FITC-QD@HMSN-PEG) and blocking group (FITC-QD@HMSN-PEG-TRC105 administrated after injection of a large dose of TRC105 antibodies). This result indicates the successful vasculature targeting and minimal nonspecific binding of QD@HMSN-PEG-TRC105 in cell culture.
In Vivo Vasculature Targeting and PET Imaging
64Cu was labeled onto NOTA-QD@HMSN-PEG-TRC105 (targeted group) and NOTA-QD@HMSN-PEG (non-targeted group) via simple mixing under mild conditions and purified with PD-10 desalting column. After radiolabeling, as-prepared 64Cu-NOTA-QD@HMSN-PEG-TRC105 (targeted group) and 64Cu-NOTA-QD@HMSN-PEG (non-targeted group) were intravenously injected into 4T1 tumor-bearing mice for in vivo vasculature targeting and PET imaging. The coronal PET images that contain the 4T1 tumors are shown in Fig. 3, and the quantitative data obtained from ROI analysis of the PET data are shown in Fig. 4.
The tumor uptake in the targeted group was prompt and persistent, manifesting as early as 3 h p.i. and remained visible after 24 h p.i. (3.1 ± 1.6, 7.2 ± 0.4, 7.2 ± 0.3, and 5.6 ± 0.3%ID/g at 0.5, 3, 6, and 24 h p.i., respectively; Fig. 4a). However, significantly lower tumor uptake was observed in non-targeted (2.7 ± 0.5, 4.6 ± 0.2, 5.0 ± 0.6, and 4.5 ± 0.7%ID/g at 0.5, 3, 6, and 24 h p.i., respectively; Fig. 4b) and blocking groups (1.4 ± 0.7, 3.9 ± 0.6, 4.9 ± 0.9, and 4.7 ± 0.7%ID/g at 0.5, 3, 6, and 24 h p.i., respectively; Fig. 4c). About 1.5-fold increase in tumor uptake was achieved with the targeted group than the non-targeted and blocking groups (p < 0.05 at 3 and 6 h p.i., respectively; Fig. 4d), suggesting the excellent targeting efficiency and specificity.
To the contrary, no significant increase from targeted group was found in other normal organs. For example, most of the NPs are eventually transported to the liver and cleared via hepatobiliary pathway [22]. In this study, slight reduction in liver uptake was observed in the targeted group (44.2 ± 9.8, 26.3 ± 3.0, 22.7 ± 2.8, and 17.2 ± 2.0%ID/g at 0.5, 3, 6, and 24 h p.i., respectively; Fig. 4a) than non-targeted (43.9 ± 4.8, 32.8 ± 4.4, 30.1 ± 4.4, and 18.8 ± 3.3%ID/g at 0.5, 3, 6, and 24 h p.i., respectively; Fig. 4b) and blocking group (58.8 ± 4.7, 31.0 ± 2.9, 26.6 ± 1.5, and 20.4 ± 0.3%ID/g at 0.5, 3, 6, and 24 h p.i., respectively; Fig. 4c), possibly because more NPs were trapped in tumor tissues in the targeted groups. In addition, the muscle which has no CD105 expression exhibited nearly identical uptakes from all three groups (targeted 0.8 ± 0.4, 1.0 ± 0.3, 0.9 ± 0.1, and 0.7 ± 0.1%ID/g; non-targeted 0.7 ± 0.1, 0.8 ± 0.1, 0.7 ± 0.1, and 0.7 ± 0.1%ID/g; blocking 0.4 ± 0.2, 0.7 ± 0.1, 0.7 ± 0.1, and 0.7 ± 0.1%ID/g at 0.5, 3, 6, and 24 h p.i., respectively), suggesting minimal nonspecific binding of 64Cu-NOTA-QD@HMSN-PEG-TRC105 to the normal organs. Furthermore, the blood uptakes from all three groups were also similar (targeted 6.7 ± 2.1, 3.3 ± 0.1, 3.3 ± 0.3, and 3.2 ± 0.4%ID/g; non-targeted 4.6 ± 4.4, 2.8 ± 0.7, 2.9 ± 0.3, and 2.8 ± 0.4%ID/g; blocking 2.9 ± 0.6, 2.9 ± 0.1, 3.2 ± 0.1, and 3.0 ± 0.2%ID/g at 0.5, 3, 6, and 24 h p.i., respectively), confirming the targeting specificity.
To further validate the accuracy of PET imaging, ex vivo biodistribution studies were conducted by wet weighting and measuring the radioactivities from tumor and other organs (Fig. 4e). The results corroborated well with the ROI analysis of PET images, where tumor uptake was significantly enhanced in the targeted group in comparison with the non-targeted and blocking groups.
Drug Loading and Optical Imaging
One of the most important advantages of HMSN over MSN is the increased drug loading capacity. Typically, due to large surface area of MSN, the anticancer drug DOX can be easily loaded onto MSN via hydrophobic and electrostatic interactions, with a loading capacity of ~ 400–500 mg g−1 (DOX weight/NP weight) [29]. After introducing a large cavity inside the MSN shells, the loading capacity was remarkably increased to 1266 mg g−1 (DOX weight/NP weight) in as-designed yolk/shell QD@HMSN NPs (Fig. 5a). The increased drug loading can potentially benefit the efficacy of cancer chemotherapy [30,31,32] and also reduce the potential in vivo cytotoxicity from the nanocarriers since smaller dose of NPs will be needed for each treatment. In addition, the drug release rate is pH dependent (Fig. 5b). In normal physiological environment (pH 7.4), 13.5 ± 0.7% of DOX was released from QD@HMSN after 48-h incubation. However, when the pH decreased to ~ 5, drug release was dramatically accelerated and 34.0 ± 0.2% of DOX was released from QD@HMSN after 48-h incubation, due to decreased hydrophobic and electrostatic interactions between DOX and silica at lower pH values. Since tumors generally have lower pH values than normal tissues, as-prepared DOX-loaded QD@HMSN can be utilized as a promising pH-sensitive drug delivery system.
Although PET imaging is highly sensitive and quantitative, it can only represent the biodistribution of radioisotopes rather than the NPs [17,18,19], since the detachment of radioisotopes and chelators from NPs is inevitable when they circulate in the blood stream, accumulate in livers and other organs, and interact with proteins and other biological macromolecules. Therefore, another imaging modality is recommended to confirm the accuracy of PET imaging and depict the real biodistribution of NPs. In this study, QD705 (Ex: 605, Em: 700 nm) inside each silica shell was employed for fluorescent imaging to understand the biodistribution of QD@HMSN NPs. In addition, anticancer drug DOX (Ex: 500, Em: 580 nm) that was loaded in the cavity of QD@HMSN NPs was also imaged to further validate their biodistribution. As shown in Fig. 5c, the tumors exhibited significant differences between targeted (NOTA-QD@HMSN(DOX)-PEG-TRC105) and non-targeted groups (NOTA-QD@HMSN(DOX)-PEG) from QD- and DOX-based optical imaging. However, no difference was found between targeted and non-targeted groups in all the normal organs. Taken together, optical imaging of QD and DOX corroborated well with the PET imaging and confirmed the successful targeting of TRC105-conjugated QD@HMSN, which can serve as a multifunctional nanoplatform for dual-modality cancer diagnosis and drug delivery.
Histological Analysis
Histological studies were conducted to evaluate the specificity of vasculature targeting. As shown in Fig. 6, excellent correlation was found between the signals from vasculature (CD 31, which is specifically expressed on vascular endothelial cells; red channel) and CD105-targeted NPs (FITC-QD@HMSN(DOX)-PEG-TRC105; green channel), indicating the excellent targeting specificity. Due to their relatively large size, minimal extravasation of NPs from the tumor vasculature was observed, which emphasizes the importance of vasculature targeting over tumor cell targeting. On the other hand, non-targeted group (FITC-QD@HMSN(DOX)-PEG) exhibited much lower tumor accumulation. Liver was selected as the positive control, since it is the major clearance organ of NPs. In our study, both targeted and non-targeted groups showed strong accumulation (green channel), and no correlation was found between the signals from vasculature and NPs in livers, suggesting that QD@HMSN NPs were captured by liver via nonspecific reticuloendothelial system (RES) uptake. In addition, minimal accumulation was found in muscle (negative control) in both targeted and non-targeted groups, which matched well with the results from PET imaging.