In Vivo Tumor-Targeted Dual-Modality PET/Optical Imaging with a Yolk/Shell-Structured Silica Nanosystem

Silica nanoparticles have been one of the most promising nanosystems for biomedical applications due to their facile surface chemistry and non-toxic nature. However, it is still challenging to effectively deliver them into tumor sites and noninvasively visualize their in vivo biodistribution with excellent sensitivity and accuracy for effective cancer diagnosis. In this study, we design a yolk/shell-structured silica nanosystem 64Cu-NOTA-QD@HMSN-PEG-TRC105, which can be employed for tumor vasculature targeting and dual-modality PET/optical imaging, leading to superior targeting specificity, excellent imaging capability and more reliable diagnostic outcomes. By combining vasculature targeting, pH-sensitive drug delivery, and dual-modality imaging into a single platform, as-designed yolk/shell-structured silica nanosystems may be employed for the future image-guided tumor-targeted drug delivery, to further enable cancer theranostics.


Introduction
In the last decade, rapid development of nanotechnology has dramatically stimulated the progress in design and synthesis of multifunctional nanosystems that can potentially be used for cancer-targeted imaging and therapy [1][2][3][4][5]. Among them, silica nanoparticles (NPs) have been one of the most widely studied nanosystems, due to their facile surface chemistry and non-toxic nature [6][7][8]. Silicabased NPs are ''generally recognized as safe'' by the United States Food and Drug Administration (FDA), highlighting their great clinical relevance [6][7][8].
Hollow mesoporous silica NPs (HMSNs), which possess numerous nanoscale pores and a large cavity inside the silica shell, have been recently developed for drug delivery due to their low mass density, large surface area, high pore volume, and uniform and tunable pore size [8][9][10]. Tumor active targeting has been applied to HMSNs in vivo to significantly enhance the tumor uptake and reduce possible side effects, leading to a desirable nanoplatform for cancer treatment [11][12][13]. Positron emission tomography (PET) has been accomplished with radiolabeled HMSNs to invasively understand their biodistribution in living animals [11][12][13]. Compared to traditional nanosystems, radiolabeled actively targeted HMSNs combine cancer diagnosis and therapy into one single platform, making cancer theranostics possible.
However, one imaging modality is not enough to accurately represent the in vivo fate of HMSNs [14][15][16]. Although PET imaging is highly quantitative and sensitive and has unlimited tissue penetration, it can only depict the in vivo biodistribution of radioisotopes, whether they are attached to or free from the carrier NPs [17][18][19][20]. Therefore, PET imaging alone may possibly lead to a totally different readout, owing to different biodistribution profiles of NP agents and free radioisotopes, which results in a false diagnosis. To overcome this drawback, we have designed a quantum dot (QD)/HMSN yolk/shell nanosystem (QD@HMSN) that makes use of the optical properties of QDs for fluorescence imaging. Compared to other fluorescent emitters such as organic dyes, QDs have wider excitation and narrower emission spectra, higher quantum yields and minimal photo bleaching, and therefore can accurately render the biodistribution of HMSNs, providing synergistic diagnostic information in addition to PET [21]. The incorporated QDs were placed in the cavity and protected by the silica shells, which will not alter the intrinsic pharmacokinetics of HMSNs in the living systems.
Unlike small molecules or antibody, NPs have a relatively large size and exhibit suboptimal extravasation from vessels, which limits their applications in tumor cell targeting. Tumor vasculature targeting avoids the need of extravasation and has become one of the most effective active targeting methods for NPs [22]. Endoglin receptor (CD105) is a type I membrane glycoprotein, which highly proliferates in tumor neovasculature and plays an important role in tumor angiogenesis, growth and metastasis [23][24][25][26]. In this study, a chimeric human/murine anti-CD105 antibody, TRC105, was conjugated onto the surface of QD@HMSN yolk/shell nanosystems for effective tumor vasculature targeting and enhanced tumor diagnosis.

Synthesis of QD@HMSN Yolk/Shell Nanosystems
QD NPs (Qdot TM 705 ITK TM carboxyl quantum dots, Thermo Fisher Scientific, Fair Lawn, NJ) were first coated with dense silica (dSiO 2 ) layer, by oil-in-water reverse micro-emulsion silica coating approach. In brief, Igepal CO-520 (NP-5, 1 mL) was dispersed in cyclohexane (20 mL) in a 100-mL three-necked flask and stirred at a slow rate for 5 min. Qdot705 (ITK organic quantum dots, 400 pmol) was added into CO-520 solution and stirred slowly for 2 h. Ammonia solution (0.14 mL, 30%) was then added into the mixture and stirred for another 2 h.
TEOS was subsequently pumped into the reaction (100 lL h -1 ) for 2 h and stirred at room temperature for 40 h. When the reaction was completed, 1 mL methanol and 2 mL ethanol were added into the solution to precipitate the sample. The QD@dSiO 2 samples were collected by centrifugation and washed with ethanol for three times. As-synthesized QD@dSiO 2 NPs were further coated with mesoporous silica NPs (MSNs) to form the mesoporous silica shell. In brief, 20 lg TEA and 10 mL CTAC (25 wt%) were dissolved into 20 mL water and stirred for 1.5 h. Ten milliliters of QD@dSiO 2 solution (containing 200 pmol QDs) was then added and stirred for another 1.5 h. TEOS was subsequently added at the rate of 20 lL min -1 for 5 min. The reaction took place at 80°C for 1 h. After the reaction was finished, QD@dSiO 2 @MSN solution was mixed with 636 mg Na 2 CO 3 and moved to 50°C water bath for 45 min to etch out dSiO 2 . The resulting QD@HMSN NPs were collected by centrifugation and washed with water for three times. To remove CTAC, the product was extracted for 24 h with a 1 wt% solution of NaCl in methanol at room temperature. This process was carried out for at least three times to ensure the complete removal of CTAC.

Surface Engineering of QD@HMSN Yolk/Shell
Nanosystems NH 2 groups were introduced onto the surface of QD@HMSN NPs for further surface engineering. QD@HMSN NPs were dispersed in 20 mL of absolute ethanol, followed by the addition of 1 mL of APTES. The system was sealed and kept at 86-90°C for reaction for 24 h. When the reaction was completed, the NH 2 -modified QD@HMSN (QD@HMSN-NH 2 ) was collected by centrifugation and washed with absolute ethanol for three times to remove the remaining APTES. For chelating radioisotopes, p-SCN-Bn-NOTA was conjugated onto QD@HMSN NPs based on the reaction between SCN and NH 2 groups at pH 9 for 2 h (QD/NOTA molar ratio 1:500; of note, since QD is the seed of HMSN, the molar concentrations of QD and HMSN NPs were nearly identical). Fluorescein isothiocyanate (FITC) was conjugated onto QD@HMSN NPs with the same method for in vitro experiments. The resulting NOTA-QD@HMSN or FITC@HMSN was then conjugated with SCM-PEG 5k -Mal based on the reaction between SCM group (succinimidyl carboxyl methyl ester) and remaining NH 2 groups at pH 7 for 2 h (QD/PEG molar ratio 1:10,000). Meanwhile, TRC105 (QD/TRC105 molar ratio 1:20) was reacted with Traut's reagent (TRC105/ Traut's reagent molar ratio 1:20) at pH 8 for 1 h to introduce free SH groups on the antibodies. Finally, as-prepared NOTA-QD@HMSN-PEG or FITC-QD@HMSN-PEG was mixed with TRC105-SH and reacted at pH 7.4 for 2 h in the presence of tris(2-carboxyethyl)phosphine (TCEP, to prevent oxidation of the thiol), generating the final products NOTA-QD@HMSN-PEG-TRC105 or FITC-QD@HMSN-PEG-TRC105 ( Fig. 1).

Cell Lines and Animal Models
All animal studies were conducted under a protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee. 4T1 murine breast cancer cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the supplier's instructions. When they reached * 80% confluence, the cells were harvested for tumor implantation. Four-to-five-week-old female Balb/c mice (Envigo, Indianapolis, IN) were each subcutaneously injected with 2 9 10 6 4T1 cells in the flank to generate the 4T1 breast cancer model. The mice were used for in vivo experiments when the tumor diameter reached 6-8 mm.

In Vitro CD105 Targeting
Human umbilical vein endothelial cells (HUVECs, CD105 positive) were harvested and suspended in cold PBS with 2% bovine serum albumin at a concentration of 5 9 10 6 cells mL -1 , incubated with FITC-QD@HMSN-PEG or FITC-QD@HMSN-PEG-TRC105 at a concentration of 10 pmol mL -1 (based on QD concentration) for 30 min at room temperature. A blocking experiment (500 lg mL -1 of TRC105 added to the cells 3 h before administration of NPs) was also carried out to further validate the targeting specificity. Afterward, the cells were collected by centrifugation at 1000 rpm for 5 min and washed three times with cold PBS. The cells were analyzed using a BD FACSCalibur 4-color analysis cytometer equipped with 488 and 633 nm lasers (Becton-Dickinson, San Jose, CA) and FlowJo analysis software (Tree Star, Inc., Ashland, OR). . Quantitative data of ROI analysis on tumor and other organs were presented as percentage injected dose per gram of tissue (%ID/g). After the last scan at 24 h p.i., mice were killed under anesthesia for ex vivo biodistribution studies. Tumor, blood, and major organs/tissues were collected and weighted. The radioactivity in the tissue was measured using a c counter (PerkinElmer) and presented as %ID/g (mean ± SD).

Drug Loading and Ex Vivo Fluorescence Imaging
Doxorubicin (DOX) was loaded into QD@HMSN yolk/ shell nanosystems as the model drug. In brief, DOX was dissolved in DMSO (10 mg  NOTA-QD@HMSN(DOX)-PEG and NOTA-QD@HMSN(DOX)-PEG-TRC105 were intravenously injected in 4T1 tumor-bearing mice. After 3 h p.i. (when 4T1 tumor uptake was at the peak based on PET imaging), mice were killed under anesthesia. Tumor, blood, and other important organs were collected for near-infrared optical imaging using IVIS spectrum in vivo imaging system (PerkinElmer, Waltham, MA). The signals from both QD (excitation/emission wavelength 605/700 nm) and DOX (excitation/emission wavelength 500/580 nm) were detected and analyzed.

Histological Analysis
Two groups of three 4T1 tumor-bearing mice were each injected with FITC-QD@HMSN-PEG and FITC- tumor, liver (positive control, which has high uptake of 64 Cu-NOTA-QD@HMSN-PEG-TRC105 based on PET imaging), and muscle (negative control, which has low uptake of 64 Cu-NOTA-QD@HMSN-PEG-TRC105 based on PET imaging) were frozen and cryo-sectioned for histological analysis. Frozen tissue slices with 6 lm thickness were fixed with cold acetone and stained for endothelial marker CD31, as described previously using a rat antimouse CD31 antibody and a Cy3-labeled donkey anti-rat IgG. All images from both FITC (representing the location of NPs in the tissues) and Cy3 (representing the location of vasculature) channels were acquired with a Nikon Eclipse Ti microscope.

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 dSiO 2 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 dSiO 2 , we can easily control the size of the hollow cavity after etching. In the second step, as-prepared QD@dSiO 2 NPs were further coated with MSN shell and then selectively etched out with Na 2 CO 3 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@dSi 2 @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 (NH 2 ) 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 64 Cu, 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.
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, asprepared 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 (E x : 605, E m : 700 nm) inside each silica shell was employed for fluorescent imaging to understand the biodistribution of QD@HMSN NPs. In addition, anticancer drug DOX (E x : 500, E m : 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.

Conclusion
Herein, we report a radiolabeled antibody-conjugated yolk/ shell-structured silica nanosystem 64 Cu-NOTA-QD@HMSN-PEG-TRC105. Significant boost of tumor uptake (1.5-fold increase at early time points) and excellent imaging contrast was achieved with specific tumor vasculature targeting. Compared to conventional MSN, the greatly enhanced drug loading capacity (1266 mg g -1 , DOX/NP weight ratio) was achieved with yolk/shell QD@HMSN NPs, which can be used for improved chemotherapy of cancer. Taking advantage of both imaging techniques, PET/optical dual-modality imaging was utilized to cross-validate the imaging results, providing a more reliable imaging outcome. By combining vasculature targeting, pH-sensitive drug delivery and dual-modality imaging into a single platform, yolk/shell-structured silica nanosystem may be employed for the future image-guided tumor-targeted drug delivery, to further enhance the therapeutic efficacy and to enable cancer theranostics.