Ultralong tumor retention of theranostic nanoparticles with short peptide-enabled active tumor homing

Lihua Li a, Yao Lu b, Zefeng Lin c, Angelina S. Mao d, Ju Jiao e, Ye Zhu f, Chunyan Jiang a, Zhongmin Yang *a, Mingying Peng *a and Chuanbin Mao *f
aState Key Laboratory of Luminescent Materials and Devices, and Guangdong Provincial Key Laboratory of Fiber Laser Materials and Applied Techniques, School of Physics, School of Materials Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510641, China. E-mail: yangzm@scut.edu.cn; pengmingying@scut.edu.cn
bDepartment of Orthopedics, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, China
cGuangdong Key Lab of Orthopedic Technology and Implant, Department of Orthopedics, Guangzhou General Hospital of Guangzhou Military Command, 111 Liuhua Road, Guangzhou, Guangdong 510010, China
dNorman North High School, 1809 Stubbeman Ave, Norman, OK 73069, USA
eDepartment of Nuclear Medicine, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510630, China
fDepartment of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, Institute for Biomedical Engineering, Science and Technology, University of Oklahoma, Norman, OK 73019, USA. E-mail: cbmao@ou.edu

Received 3rd January 2019 , Accepted 17th May 2019

First published on 17th June 2019


Computer tomography (CT) and magnetic resonance imaging (MRI) are noninvasive cancer imaging methods in clinics. Hence, a material that enables MRI/CT dual-modal imaging-guided therapy is in high demand. Currently, the available materials lack active tumor targeting, deep tumor penetration, and ultralong tumor retention and may lose their imaging elements. To overcome these drawbacks, herein, nanoparticles (NPs) were developed by integrating an MRI contrast-enhancing chelated gadolinium (Gd) complex within a doxorubicin (DOX)-loaded protective silica shell as well as a CT imaging/photothermal biocompatible bismuth (Bi) nano-core, which surface-displayed an MCF-7 breast tumor-homing peptide (AREYGTRFSLIGGYR, termed AR); we found that the resultant NPs AR-Bi@SiO2-Gd/DOX could home to and penetrate deep into the tumors with the unexpected ultralong retention of at least 14 days (as determined by CT/MRI imaging) and the tumor retention half-life of 104.5 h (as determined by ICP-MS analysis) under the guidance of the AR peptide. These NPs can be further used to image tumors with significantly increased sharp contrasts via both CT and MRI, which are much better than the commercial standard contrast agents; moreover, they significantly inhibit tumor growth via the synergistic action of both Bi-enabled photothermal therapy and DOX-induced chemotherapy. The NPs are cleared by the spleen, liver and kidney and then excreted from the body along with faeces and urine. The precise tumor targeting and ultralong tumor retention of these unique NPs would enable both precise tumor detection for early diagnosis and signal-persistent tumor tracking for monitoring the treatment with only a single injection of these NPs.



New concepts

When a drug is applied to treat tumors, ideally, the tumors should be monitored by clinically available, minimally invasive imaging techniques, such as CT and MRI, using the same drug as an imaging probe. Therefore, a tumor-homing theranostic nanoparticle (NP) is needed to serve as both a drug and a CT/MRI dual-imaging probe. Moreover, to ensure that the same (and only one) dose of the NP is used first for treating the tumors and then for monitoring/tracking them post-treatment, the NP should be retained in the tumors for a long time. However, to date, although some reported NPs present the tumor retention time of over 2 days, their imaging signals significantly decrease over time. In our study, our uniquely designed theranostic NPs demonstrated the record ultralong tumor imaging time of >14 days. Herein, the tumor-homing peptide-modified Bi@SiO2-Gd NPs could home to and penetrate deep into the tumors with the unexpected ultralong retention time of at least 14 days, leading to targeted tumor destruction and sustained CT/MRI imaging contrast enhancement. This study indicates that short tumor-homing peptides can enable the theranostic agents to achieve ultralong tumor retention and become powerful in treating tumors and monitoring them post-treatment using only one injection.

Introduction

The integration of multimodal imaging and therapeutic agents into one theranostic platform holds significant promise for personalized medicine. Normally, this platform can be achieved by mixing different inorganic elements,1 conjugating organic and inorganic agents,2 or developing all-in-one nanoparticles (NPs) with multimodal therapeutic and imaging abilities.3 X-ray computed tomography (CT) and magnetic resonance imaging (MRI) have played a significant role in non-invasive tumor diagnosis in clinics because CT can offer 3D reconstruction of the regions of interest in deep hard tissues, whereas MRI can provide multi-parameter and azimuth imaging in deep soft tissues. In addition to their unique advantages, their shortcomings, including inadequate sensitivity, poor spatial resolution and single modality, limit their applications in accurate clinical information collection and early tumor diagnosis. Due to the significant development of nanotechnology, various contrast agents have been developed to enhance the image contrast and provide more precise functional information; moreover, multimodal imaging contrast nanoagents, which can enhance image contrast via different techniques, have been developed that will improve the accuracy and sensitivity of disease diagnosis.4–6 For example, gadolinium (Gd) or iron oxide as an MRI contrast agent and gold as a CT contrast imaging agent have been combined to develop multimodal imaging materials.7–9

Recently, bismuth (Bi)-doped NPs have attracted the attention of researchers for enhancing the CT imaging contrast because Bi, as a high atomic number element, has large X-ray attenuation capability and outstanding biocompatibility;10–12 especially, Bi, an element that is friendly to the human body13 either in the pure elemental form or in the compound form, can strongly absorb near infrared (NIR) light and then convert the NIR light energy into heat; thus, it acts as a photothermal agent.11,14 Hence, the integration of Bi and Gd into one system is a promising approach to develop a NP that can serve not only as a photothermal therapy agent but also as a CT and MRI dual-modal imaging probe; however, this is very difficult to achieve. Thus, very few studies have been dedicated in this direction. Very recently, Bi and Gd NPs were synthesized for achieving a dual-modal imaging-guided radiation therapy.15 However, the resultant Gd-Bi systems lacked a proper Bi/Gd ratio (resulting in negligible CT contrast imaging) and tumor-homing capability and could not be easily functionalized and loaded with anti-cancer drugs. More importantly, the currently used nanotheranostics lack active targeting, deep tumor penetration, and long tumor retention (typically for only up to 24–48 h),16 resulting in side effects and ineffective tumor imaging and therapy. In addition, when the NPs are not modified by a tumor-homing molecule, they face limitations in tumor imaging. For example, they cannot be retained in tumors and thus accumulate in the lymph nodes18 and have to be injected intratumorally to ensure tumor accumulation;19 moreover, tumor imaging is achieved only when NP-labeled cancer cells are injected to generate tumor models,20 or these NPs only have single-modal imaging (e.g., fluorescence imaging) with no therapeutic function.21 Thus, they are not ideal nanotheranostics due to the lack of tumor-homing capability. Although antibodies may be used to modify nanotheranostics, they are more expensive and immunogenic than small peptides.17 Hence, small peptides are highly desired for enabling the nanotheranostics to exhibit tumor targeting capability.

To overcome the abovementioned daunting challenges, we designed a Gd-Bi nanocomposite particle composed of a spherical Bi NP core and a silica shell loaded with and protecting a complex of gadolinium and diethylenetriamine pentaacetic acid (Gd-DTPA) (Scheme 1). The resultant NP is yolk-like and termed Bi@SiO2-Gd. Using an in vivo phage display technique, from a phage-displayed random peptide library, we recently discovered and validated a peptide with the sequence of AREYGTRFSLIGGYR (termed AR) that could selectively home to the MCF-7 breast tumor.22 The AR peptide was conjugated to the surface of carboxylated Bi@SiO2-Gd to make the NPs capable of actively targeting the MCF-7 breast tumor. The resultant NPs, termed AR-Bi@SiO2-Gd NPs, were then allowed to carry doxorubicin (DOX) to form AR-Bi@SiO2-Gd/DOX due to their porous structure. The resultant AR-Bi@SiO2-Gd/DOX NPs were administered to mice bearing the MCF-7 breast tumor by intravenous (i.v.) injection. We found that the AR-Bi@SiO2-Gd/DOX NPs efficiently homed to and penetrated deep into the tumor (with the ultra-long retention time of at least 14 days); moreover, they functioned not only as CT and MRI dual-modal imaging contrast agents for precise deep tissue cancer diagnosis but also as synergistic photothermal and chemo-therapeutic agents for efficiently destroying the tumor (Scheme 1).


image file: c9mh00014c-s1.tif
Scheme 1 Schematic of the AR-Bi@SiO2-Gd/DOX NPs in tumor-targeting CT/MRI dual-modal imaging and synergistic photothermal-chemotherapy.

Results and discussion

Synthesis and characterization of AR-Bi@SiO2-Gd

To synthesize the AR-Bi@SiO2-Gd/DOX NPs (Fig. 1a), we first chemically synthesized spherical Bi NPs (70–75 nm) with a rhombohedral crystal structure (Fig. 1b and Fig. S1, ESI). Then, the hydrophilic Bi@SiO2 NPs were first fabricated by employing a reverse-microemulsion method23 (Fig. 1c). We further loaded the Gd-DTPA complexes (a probe for MRI) onto the surface of Bi@SiO2, followed by the deposition of a porous silica shell again to form Bi@SiO2-Gd NPs with a unique yolk-like structure (130 nm, Fig. 1d). High-angle annular dark-field scanning TEM (HAADF-STEM) was performed to map the elemental distribution of Gd, Bi and Si elements in the NPs and thus confirmed the yolk-like structure of the Bi@SiO2-Gd NPs (Fig. 1e).
image file: c9mh00014c-f1.tif
Fig. 1 Design, fabrication and properties of the AR-Bi@SiO2-Gd/DOX NPs. (a) Schematic of the fabrication of AR-Bi@SiO2-Gd/DOX NPs with highly effective MCF-7 breast tumor targeting and drug loading capability. (b–e) The TEM images of Bi NPs (b), Bi@SiO2 NPs (c), and AR-Bi@SiO2-Gd NPs (d) as well as the STEM image and Si, Gd, and Bi mapping of the NPs (e). Scale bar = 50 nm. (f) Absorption spectra of Bi@SiO2-Gd, AR-Bi@SiO2-Gd, ddH2O and free AR peptide. (g) Quantitative temperature changes of NPs versus NP concentrations under 808 nm irradiation (1 W cm−2). (h) Heating/cooling experiment of Bi@SiO2-Gd (125 μg mL−1) under 808 nm laser irradiation (1 W cm−2), the laser was kept on from 0 to 600 s and then remained off from 600 to 1000 s. The photothermal conversion (PTC) efficiency (η) was calculated to be 28.47%. (i) FTIR spectra of Bi, Bi@SiO2, Bi@SiO2-Gd, AR-Bi@SiO2-Gd and free AR peptide. The band between 3000 and 4000 cm−1 in the free AR is due to the presence of water. (j) DOX loading efficiency on AR-Bi@SiO2-Gd. (k) DOX release from AR-Bi@SiO2-Gd/DOX under acidic (pH 5) and neutral (pH 7.4) conditions and at low (room temperature) and body (37 °C) temperature.

The porous structure of the Bi@SiO2-Gd NPs was further investigated by the Brunauer–Emmett–Teller (BET) analysis. We found that the NPs presented a higher specific surface area (218.778 cm2 g−1) as well as the average pore volume of 0.2 cm3 g−1 and the pore size of 4.5 nm (Fig. S3, ESI). The unique structure can ensure the full contact of the contrast agent with water while preventing the leakage of Gd3+ since the chelates in Gd-DTPA may form hydrogen bonds with the hydroxyl groups in silica.24,25 The mass ratio of Bi and Gd in the NPs was determined to be 2.15[thin space (1/6-em)]:[thin space (1/6-em)]1 by a further inductively coupled plasma mass spectrometry (ICP-MS) analysis (Gd and Bi account for 13.08% and 28.13% by weight in the AR-Bi@SiO2-Gd/DOX, respectively). The mean hydrodynamic diameter of the Bi@SiO2-Gd NPs was 130–190 nm, as determined by dynamic light scattering (DLS) measurement (Fig. S2a, ESI), consistent with the size revealed by TEM imaging.

The Bi@SiO2-Gd NPs were subsequently modified first with 3-aminopropyltriethoxysilane (APTES) to form Bi@SiO2-Gd-NH2 and then with succinic anhydride (SA) to form carboxylated Bi@SiO2-Gd (Bi@SiO2-Gd-COOH), which was further cross-linked with the AR peptide via a typical N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) method to form the AR-Bi@SiO2-Gd NPs. Note that the C-terminal end of the AR peptide was added with an NH2-rich linker (GGGGKKKK) to facilitate the cross-linking of the AR peptide with Bi@SiO2-Gd-COOH; this enabled the AR peptide to protrude from the resultant NP surface for tumor homing. The zeta potential of the NPs after AR peptide conjugation was increased but remained negative overall (Fig. S2b, ESI); this confirmed successful conjugation of the cationic peptide. The Bi@SiO2-Gd NPs before and after peptide conjugation presented a similar broad NIR absorption band (Fig. 1f). In addition, the zeta potential of CP-Bi@SiO2-Gd and PEG-Bi@SiO2-Gd has changed when compared with that of Bi@SiO2-Gd-COOH; this indicates the successful conjugation of the peptide (Fig. S2b, ESI). Moreover, the Bi@SiO2-Gd NPs with AR conjugation exhibited a new absorption peak at 270 nm as compared to those without AR conjugation; this indicated the successful conjugation of the AR peptide into the NPs. The successful conjugation of AR into the Bi@SiO2-Gd NPs was also verified by Fourier transform infrared (FTIR) spectroscopy (Fig. 1i). The amount of the AR peptide conjugated into the Bi@SiO2-Gd NPs was determined to be 23.5 μg mg−1 by the bicinchoninic acid (BCA) method. The resultant AR-Bi@SiO2-Gd NPs were found to be non-cytotoxic and would not harm the blood (even when the NP concentration reached the high level of 500 μg mL−1), as observed via a cytotoxicity and hemolytic assay (Fig. S4, ESI).

Photothermal and drug loading/release behaviors of the AR-Bi@SiO2-Gd NPs

The AR-Bi@SiO2-Gd NPs exhibited a concentration-dependent photothermal response under the 808 nm NIR irradiation with the power density of 1 W cm−2 (Fig. 1g and h). The temperature of the NPs (0.5 mg mL−1) was increased from 25 °C to 68.9 °C after continuous irradiation. The NP solution could reach the temperature of up to 48.1 °C even at 125 μg mL−1. However, the temperature of pure water could only reach 30 °C when the same irradiation was applied to it. The photothermal conversion efficiency (η) was determined to be 28.47% using a reported method (Fig. S5, ESI).14 These results indicated that the NPs could be used as a therapeutic agent in the NIR-triggered photothermal therapy.

The AR-Bi@SiO2-Gd NPs were then allowed to interact with DOX to achieve the loading of the cationic DOX via its electrostatic interaction with the anionic silica shell on the NPs; this resulted in the formation of the AR-Bi@SiO2-Gd/DOX NPs. After centrifugation, the AR-Bi@SiO2-Gd/DOX NPs exhibited enhanced absorption at 482 nm, and the supernatant showed significantly reduced absorption at the same position as compared to the initial DOX solution (Fig. 1j); this confirmed the loading of DOX onto the NPs after incubation for 12 h. The encapsulation efficiency was thus calculated to be 54.5%, and the loading content was 54.56 μg mg−1, suggesting highly efficient drug loading onto the AR-Bi@SiO2-Gd/DOX NPs. A similar loading behavior was found on the PEG-Bi@SiO2-Gd/DOX NPs (Fig. S6, ESI). We found that DOX was preferentially released from the NPs at both room and body temperature when the media was acidic (representing the acidic extracellular and intracellular environments in the tumor) (Fig. 1k).26,27 In addition, the drug release was evaluated during and after irradiation (Fig. S7, ESI). A burst DOX release was found in the pH = 5 PBS solution during irradiation; then, the release behavior became gentle. This pH- and NIR-responsive drug release ensures that DOX would be preferentially released in the tumors after the targeting peptide guides the NPs to arrive at the tumor site. The pH-responsive DOX release might arise from the increased solubility of electrostatically adsorbed DOX in the media under acidic conditions.28

In vitro targeted cancer therapy

To verify the capability of AR targeting, we used the same methods to prepare control NPs, where AR was replaced by its scrambled version (i.e., the control peptide with the sequence of EARGFSLGYRIYGRT, termed CP), or polyethylene glycol (PEG). The CP and PEG-modified NPs were termed CP-Bi@SiO2-Gd/DOX and PEG-Bi@SiO2-Gd/DOX, respectively. An in vitro study using these NPs confirmed that the AR peptide enabled the NPs to specifically home to the MCF-7 breast cancer cells (Fig. 2 and Fig. S8, ESI) rather than to other control cells (Fig. S9, ESI), and this excellent targeting led to the selective killing of MCF-7 breast cancer cells (instead of the normal breast cancer cells MCF-10A) by the AR-Bi@SiO2-Gd/DOX NPs via synergistic photothermal and chemotherapy (Fig. 2, Fig. S10 and S11, ESI).
image file: c9mh00014c-f2.tif
Fig. 2 More effective in vitro targeted killing of MCF-7 cancer cells by the AR-Bi@SiO2-Gd/DOX NPs under NIR. (a) Confocal fluorescence images of the MCF-7 cells co-cultured with FITC-labeled NPs, including FITC-labeled PEG-Bi@SiO2-Gd, CP-Bi@SiO2-Gd and AR-Bi@SiO2-Gd NPs. The cell nuclei were dyed with Hoechst 33342. The FITC-labeled AR-Bi@SiO2-Gd NPs were found to enter both the cell cytoplasm and the nucleus. (b) Flow cytometry analysis of the MCF-7 cells stained by the free dye molecules (FITC) and dye-labeled NPs, including the FITC-labeled PEG-Bi@SiO2-Gd, CP-Bi@SiO2-Gd and AR-Bi@SiO2-Gd NPs. In the blocking group, the free AR peptide was incubated with the MCF-7 cells, followed by the addition of FITC-labeled AR-Bi@SiO2-Gd NPs. (c) Fluorescence intensity of the MCF-7 cells stained by the free dye molecules (FITC) and dye-labeled NPs. (d) The in vitro combined photothermal-chemo killing effect of PBS, DOX, and DOX-loaded NPs with or without 808 nm irradiation for 5 min, suggesting that AR-Bi@SiO2-Gd/DOX is most effective in killing cancer cells under NIR. (e) Live/dead assay of the MCF-7 cells after being co-cultured with different groups, indicating that the AR-Bi@SiO2-Gd/DOX + NIR group could kill the cancer cells most effectively. Scale bar = 50 μm. *p < 0.05, **p < 0.01.

Specifically, to study the in vitro cancer targeting, we used FITC to fluorescently label the AR-Bi@SiO2-Gd, CP-Bi@SiO2-Gd and PEG-Bi@SiO2-Gd NPs. After the MCF-7 cells were allowed to interact with different NPs in the culture medium and washed to remove the non-binding NPs, the cells were fluorescently imaged. The cells in the FITC-labeled AR-Bi@SiO2-Gd NPs group exhibited strongest fluorescence among all groups (Fig. 2a). Moreover, the confocal images showed that the AR-modified NPs could enter both the cytoplasm and the cell nucleus. However, for the FITC-labeled CP-Bi@SiO2-Gd and PEG-Bi@SiO2-Gd groups, much fewer NPs entered the cancer cells, and a similar but weak green fluorescence was detected from the cells (Fig. 2a and Fig. S9, ESI). The results of the flow cytometry analysis of the cells (Fig. 2b and c) agreed well with the fluorescence imaging results of the cells (Fig. 2a); this further proved the excellent tumor-targeting capability of AR-Bi@SiO2-Gd. Interestingly, when the FITC-labeled AR-Bi@SiO2-Gd NPs were incubated with normal breast cells (MCF-10A) as well as other types of breast cancer cells (SKBR-3 cells and MDA-231 cells) (Fig. S9, ESI), the fluorescence imaging almost detected no fluorescence from these control cells; this confirmed the excellent MCF-7 tumor homing specificity of the AR-modified NPs. Moreover, in the blocking group, the AR-modified NPs were allowed to interact with the MCF-7 cells after the cells were incubated with free AR peptides. We found that the AR-modified NPs could not target the AR peptide-blocked MCF-7 cells; this confirmed that the AR peptide could specifically target the MCF-7 cells.

We then investigated the synergistic photothermal and chemotherapy in vitro by the AR-Bi@SiO2-Gd/DOX NPs. Different NP groups were used to treat the MCF-7 cells under NIR irradiation (0 or 1 W cm−2). At first, the MTT assay was conducted on the normal breast cells MCF-10A (Fig. S10, ESI). It showed that more than 80% cells were still alive even at the NP concentration of 500 μg mL−1; this illustrated excellent biocompatibility of the NPs. The MTT assay results (Fig. 2d) indicated that the application of only NIR or NPs caused almost no influence on the cancer cells, whereas the AR-Bi@SiO2-Gd + NIR group dramatically reduced the cell survival rate to 30% due to the hyperthermia effect. Note that fewer than 20% cells survived in the AR-Bi@SiO2-Gd/DOX + NIR group; this confirmed the significantly enhanced cell killing effect of the synergistic therapy. Moreover, the fluorescence live/dead staining was conducted to investigate the synergistic therapy. All cancer cells became round and red in the AR-Bi@SiO2-Gd/DOX + NIR group (Fig. 2e); this indicated that the MCF-7 cancer cells were suffering from irreversible damage due to the targeted combined photothermal and chemo-therapy. Moreover, when the AR peptide was replaced with PEG or CP, the cancer cell killing capability was significantly reduced (Fig. 2d); this further confirmed the MCF-7 tumor targeting capability of the AR-Bi@SiO2-Gd/DOX NPs. In addition, when no DOX was loaded into the NPs or when no NIR was applied to the DOX-loaded NPs, the AR-Bi@SiO2-Gd/DOX NPs did not show effective cancer cell killing (Fig. 2d); this suggested the synergistic photothermal-chemo therapy enabled by the AR-Bi@SiO2-Gd/DOX NPs. Interestingly, when the NPs loaded with or without DOX were co-cultured with normal breast cells (MCF-10A) and then carefully washed off, all the cells were in good conditions (Fig. S10 and S11, ESI) due to the outstanding biocompatibility of NPs and the inhibited DOX drug release in the neutral medium. These results confirmed that our NPs enhanced the targeted in vitro photothermal-chemotherapy.

CT and MRI contrast enhanced by NPs

We continued to examine the in vitro use of Bi@SiO2-Gd to enhance the CT imaging contrast as compared to that in the case of a commercial CT contrast agent (iobitridol). The Hounsfield unit (HU) values were increased when the concentrations of the contrast agent were increased from 0 to 25 mg mL−1 at 80 kV. A significant enhancement of HU was observed in Bi@SiO2-Gd (slope, 31.56) as compared to that in the case of iobitridol (slope, 24.12) at the same concentration (Fig. 3a) because Bi could strongly attenuate X-ray29,30 and was present in the Bi@SiO2-Gd NPs in high amount. After different NPs were i.v. injected into the tumor-bearing mice, the tumors could be clearly visualized and detected by CT imaging in the AR-Bi@SiO2-Gd group (Fig. 3c and Fig. S12, ESI), whereas they could only be weakly imaged in the CP-Bi@SiO2-Gd NPs and could not be detected in the PEG-Bi@SiO2-Gd NPs (Fig. 3c). The latter two control NPs showed significantly weaker and even no contrast in tumor imaging because they reached the tumors, although in a very limited amount, via an inherent tumor property termed enhanced permeability and retention (EPR) (Fig. 3c). However, the AR-Bi@SiO2-Gd NPs efficiently reached the center of the tumors due to their outstanding tumor homing capability and subsequent high accumulation at the tumor site within 24 h. Moreover, the CT contrast signal at the tumor site from the AR-Bi@SiO2-Gd NPs was gradually enhanced in the first 24 h (Fig. 4c and Fig. S12, ESI). We further found that the AR-Bi@SiO2-Gd NPs could still be effectively enriched at the tumor site and enhance the CT contrast 7 days post-injection (Fig. 4a) probably because the NPs could be effectively trapped in the tortuous tumor vessels due to the AR guiding. Moreover, defects in the lymphatic drainage system can help capture the NPs and delay their removal,31,32 resulting in the accumulation of NPs in the tumor and strong long-term CT imaging contrast enhancement. To the best of our knowledge, a CT agent with this ultralong retention time has never been reported. Currently, most theranostic NPs are found to only enhance the tumor signals within 1–2 days.33
image file: c9mh00014c-f3.tif
Fig. 3 Tumor detection by CT imaging and T1-weighted MRI enabled by AR-Bi@SiO2-Gd. (a) In vitro CT imaging enhanced by Bi@SiO2-Gd at different concentrations. The CT value (HU) of Bi@SiO2-Gd and iobitridol with different concentrations at 80 kV. The slope of Bi@SiO2-Gd is 30.39, whereas for commercial iobitridol, it is 24.35. (b) In vitro T1-weighted MRI imaging enhanced by Bi@SiO2-Gd with different Gd concentrations. The linear curve of relaxation rate (1/T1) versus the Gd3+ concentration in the standard Gd-DTPA and Bi@SiO2-Gd solution. The r1 for Bi@SiO2-Gd and Gd-DTPA is 16.89 and 4.1 mM−1 s−1, respectively. (c) The CT images of the MCF-7 tumor-bearing nude mice obtained at 3 and 24 h post-injection of 200 μL PEG-Bi@SiO2-Gd, CP-Bi@SiO2-Gd and AR-Bi@SiO2-Gd NPs (5 mg kg−1, i.e., with the dose of 5 mg per kg of mouse). (d) In vivo coronal T1-weighted MRI images of tumor-bearing mice before and after injection of 200 μL NPs (5 mg kg−1). For each panel shown in (d), the top and bottom images are the grey scale and the corresponding pseudo-colored images, respectively. Each tumor site is highlighted by a circle in both (c) and (d). These data suggest that the tumor targeting of AR-Bi@SiO2-Gd NPs resulted in the enrichment of NPs in the tumors, making it possible to precisely detect tumors by CT and MRI.

image file: c9mh00014c-f4.tif
Fig. 4 Strong CT/MRI contrast enhancement and ultra-long tumor retention of AR-Bi@SiO2-Gd NPs. (a and b) CT contrast images (a) and coronal T1-weighted MRI images (b) showing the detection of the tumors, 7 days after the MCF-7 bearing mice were intravenously injected with 200 μL PEG-Bi@SiO2-Gd, CP-Bi@SiO2-Gd and AR-Bi@SiO2-Gd NPs (5 mg kg−1) through a tail vein. In (b), the top and bottom images are the grey scale and the corresponding pseudo-colored images, respectively. The tumors in (a) and (b) are highlighted by red circles. (c) Changes in the CT contrast (left) and the signal-to-noise ratio obtained from the T1-weighted MRI images (right) at the tumor site. The results indicate that the AR-Bi@SiO2-Gd NPs can still contribute to contrast enhancement at the tumor sites for more than 7 days post injection. *p < 0.05, **p < 0.01.

We further investigated the capability of the Bi@SiO2-Gd NPs in enhancing the MR imaging contrast. The longitudinal proton relaxation time (T1) of the NPs and commercial Gd-DTPA was studied in parallel using a 3 T MRI scanner. The longitudinal relaxivity of the Bi@SiO2-Gd NPs was 16.89 mM−1 s−1 (Fig. 3b), 4-fold higher than that of Gd-DTPA (r1 = 4.1 mM−1 s−1). This suggested that our NPs showed higher capability of enhancing the MRI contrast than the commercial contrast agent. We further tested the precise tumor targeting capability of the AR-Bi@SiO2-Gd, CP-Bi@SiO2-Gd and PEG-Bi@SiO2-Gd NPs injected into the breast tumor-bearing mice (Fig. 3d). After 3 h of i.v. injection, clearly enhanced T1-weighted signals were detected at the tumor site for the AR-Bi@SiO2-Gd group as compared to those for the other non-targeting groups. Moreover, the enhanced MR signals at the tumor sites became stronger one day post-injection and remained sufficiently strong even 7 days post-injection (Fig. 4d), as judged by the brighter contrast at the tumor sites for the AR-Bi@SiO2-Gd group. Both the MRI and the CT imaging suggest that due to their precise tumor targeting, deep tumor penetration and ultralong tumor retention, the AR-Bi@SiO2-Gd NPs will enable early tumor detection and long-term tumor monitoring during treatment after only a single dose.

In addition, the early detection and long-term monitoring (during tumor treatment by AR-Bi@SiO2-Gd/DOX + NIR) of the tumors were studied (Fig. S13, ESI). The enhancement of the CT and T1 signals was clear 1 day post treatment. With the atrophy of the tumors due to the treatment, the CT/MR signals were decreased over time; this illustrated the real-time tumor tracking ability of the AR-Bi@SiO2-Gd NPs during the treatment. The CT/MR signal enhancement over the ultralong period of at least 14 days without any treatment was also evaluated (Fig. S14, ESI). We found that there was still some CT/MR contrast enhancement even 14 days after the single injection although the contrast on day 14 was significantly lower than that on day 10. These data further suggested that our NPs could be used to track tumors for a long period of time.

In vivo tumor-targeted photothermal and chemo-therapy by NPs

Encouraged by the enhanced CT/MRI imaging, excellent tumor targeting, high drug loading and efficient photothermal conversion, we evaluated the therapeutic use of these NPs in the treatment of the MCF-7 tumors after i.v. injection into the tumor-bearing mice. When the tumors grew to the size of 75–100 mm3, the animals were randomly divided into 7 groups with different treatments. At 3 h after the i.v. injection of different NPs, NIR irradiation was applied to the tumor to trigger the photothermal therapy. The highest temperature in the tumor center for the AR-Bi@SiO2-Gd group reached 47.9 °C (Fig. 5a and Fig. S15, ESI), which was above the minimum temperature (∼41 °C) needed to cause an irreversible damage to the tumor tissues.34,35 The highest temperature in the tumor center for the PEG-Bi@SiO2-Gd and CP-Bi@SiO2-Gd groups could also reach around 41 °C due to the EPR effect (Fig. 5a and Fig. S15, ESI). In contrast, only mild temperature changes were observed for the NaCl + NIR group. The inhibition of tumor growth was most significant in the AR-Bi@SiO2-Gd/DOX + NIR group among all groups due to the presence of the tumor-homing AR peptide and the synergistic photothermal36 and chemo-therapy (Fig. 5b–d). When no NIR was applied or no drug was loaded, the corresponding groups AR-Bi@SiO2-Gd/DOX or AR-Bi@SiO2-Gd + NIR showed reduced tumor inhibition (Fig. 5d). The tumors in the DOX, AR-Bi@SiO2-Gd/DOX and other control groups grew quickly with the same trend. These results indicated negligible tumor inhibition effect with the injection of only DOX or AR-Bi@SiO2-Gd/DOX. The tumors were suppressed in the first 7 days in the AR-Bi@SiO2-Gd + NIR group due to the hyperthermia effect, but still presented a growth trend in the next 7 days (due to the lack of DOX toxicity). Moreover, the tumors were not completely suppressed in the PEG-Bi@SiO2-Gd/DOX + NIR and CP-Bi@SiO2-Gd/DOX + NIR groups due to insufficient NP enrichment. To further validate the targeted cancer therapy, we acquired the tumors of different treatment groups (on day 14) and characterized them by H&E staining for histopathological analysis (Fig. 5e). The cells in the AR-Bi@SiO2-Gd/DOX + NIR group suffered from severe necrosis with a small round shape, whereas only partial damages were found in the AR-Bi@SiO2-Gd + NIR group. The hyperthermia at the tumor site made the cancer cells more sensitive to DOX and induced dual-modal destruction in the tumors. The animal weight change during the tumor treatment did not show significant changes among all groups (Fig. 5e); this suggested the minimum side effects of our NPs. Thus, the AR-Bi@SiO2-Gd/DOX NPs are promising breast cancer targeted therapeutics.
image file: c9mh00014c-f5.tif
Fig. 5 The in vivo tumor-targeted cancer therapy by the AR-Bi@SiO2-Gd/DOX + NIR group via synergistic photothermal and chemo-therapy. (a) IR images of the animals treated with different groups under 808 nm irradiation (1 W cm−2) for 5 min. (b) Typical images of the MCF-7 tumor-bearing mice 14 days after treatment in different groups: (i) AR-Bi@SiO2-Gd/DOX + NIR; (ii) AR-Bi@SiO2-Gd + NIR; (iii) CP-Bi@SiO2-Gd/DOX + NIR; (iv) AR-Bi@SiO2-Gd/DOX; (v) PEG-Bi@SiO2-Gd/DOX + NIR; (vi) DOX + NIR; (vii) control. The tumors are highlighted with red circles. (c) Typical images of tumors harvested from different treatment groups in (b). (d) Tumor volume change in different treatment groups. (e) Body weight of the mice during treatment. (f) H&E stained images of tumors in different treatment groups in (b). Scale bar = 200 μm. These data confirm that the AR-Bi@SiO2-Gd/DOX outperform other NPs by selectively accumulating in the tumors and destroying them by synergistic photothermal-chemotherapy under NIR. *p < 0.05, **p < 0.01.

The ultralong tumor retention of NPs

The AR-Bi@SiO2-Gd NPs exhibited the relatively long tumor retention half-life of 104.5 h (Fig. S16, ESI). This long retention may be due to the binding of the peptide to the cancer cells in the tumor, high tumor vessel leakage, and the proper size of the NPs (<200 nm). Commonly, the size of the gaps in the tumor vasculature ranges from 100 to 800 nm,37,38 and particles smaller than 200 nm have been found to more effectively pass through the gaps.39 Currently, the reported tumor retention time for inorganic theranostic NPs is generally between 6 h and 48 h.9,15,16 For example, SiBiGd NPs have the tumor retention time of 1 h; this makes it impossible to image them after 0.5 h.15 In another example, the newly reported Gd-PEG-Bi NPs have the long MRI imaging time of 24 h at the tumor site due to their relatively long blood circulation and tumor retention time; however, the CT imaging contrast becomes very weak even after 3 h.16 These results showed that our AR-Bi@SiO2-Gd NPs exhibited the longer tumor retention time and stronger CT imaging contrast than the reported inorganic NPs. Therefore, our tumor-homing peptide has significantly enhanced the tumor retention time. In addition, it is likely that the retention time is highly dependent on the particle size.40 The ultralong retention might also be partially contributed by the NP-induced endothelial leakiness.41–44

Biosafety, metabolism and clearance of NPs

Biosafety is vital for the further clinical application of NPs. The H&E staining of the main organs and the distribution of Bi/Gd were analyzed at 3 h, 24 h, 48 h and 168 h post injection of the NPs. There was no adverse effect or tissue damage in the main organs after different treatments (Fig. S17, ESI). The myocardial cells and glomerulus in the various treatment groups were intact and clear, the hepatocytes and splenocytes were normal, and no obvious damages were identified in the main organs. These observations indicated the outstanding biocompatibility of the NPs. To investigate the metabolism and clearance of the NPs, we first compared the Bi and Gd biodistribution in different NPs groups (CP-Bi@SiO2-Gd, AR-Bi@SiO2-Gd and PEG-Bi@SiO2-Gd) one day post injection. The AR-Bi@SiO2-Gd NPs were found to effectively accumulate at the tumor sites; on the other hand, their decreased accumulation was observed in the liver as compared to that for the PEG-Bi@SiO2-Gd group (Fig. S18, ESI). We then investigated the metabolism of AR-Bi@SiO2-Gd NPs at 3, 24, 48 and 168 h post i.v. injection (Fig. S19 and S20, ESI). We found that the NPs presented appreciable accumulation in the liver and spleen probably due to phagocytosis of the reticuloendothelial system. The Bi and Gd contents at the tumor sites were also higher than those in the peritumoral tissues from 3 to 168 h. Interestingly, we found that more than 40% residual Bi and Gd were still at the tumor site; however, the amount of Bi and Gd was significantly decreased (with the residual being less than 20%) in other main organs even after a week; this was consistent with the enhanced targeted CT imaging effect of the AR-Bi@SiO2-Gd NPs (Fig. 3 and 4). In addition, we further investigated the effects of the AR-Bi@SiO2-Gd NPs on the blood routine variables as well as the liver and kidney functions in mice by a blood test. We found that there were no significant differences between the treatment group and control group (NaCl injection); this illustrated the excellent in vivo biocompatibility of the synthesized NPs (Fig. S21, ESI).

The metabolism and clearance of AR-Bi@SiO2-Gd NPs were further analysed by determining the content of Gd and Bi in the blood, urine and faeces (Fig. S22 and S23, ESI). It was found that Bi and Gd were present in high contents in the urine and faeces in the first 2 days. The metabolism peak in the faeces occurred within the first 3 h, whereas the metabolism peak in the urine attained the strongest signal at 24 h. The Bi and Gd content decreased significantly with time. After 7 days, the Bi and Gd content in the main organs (except tumors, blood and metabolites) decreased significantly, but still presented high level at the tumor site (this was consistent with the observed ultralong tumor retention). These results suggested a possible metabolic pathway of the AR-Bi@SiO2-Gd NPs; the NPs in the blood circulation were cleared by the spleen, liver and kidney and then excreted from the body along with the faeces and urine. The leakage of Gd3+ during the AR conjugation, drug loading and release process (Fig. S24, ESI) and in different solutions was also tested in 30 days. The results showed that no more than 0.5% Gd was leaked from the NPs, further illustrating the biosafety of our NPs and their potential applications as theranostic agents (Fig. S25, ESI).

Overall, we verified that the AR-Bi@SiO2-Gd/DOX NPs were enriched at the tumor sites than in the peritumoral tissues (Fig. S16, ESI), presented decreased accumulation in the liver as compared to other control NPs and thus had no adverse effects after treatments. A clearance study by the analysis of the Bi and Gd content in the blood, urine and faeces (Fig. S20 and S21, ESI) suggested that the NPs in the blood circulation could be cleared from the body. These results suggest that our AR-Bi@SiO2-Gd NPs are potentially safe for breast cancer theranostics.

Conclusions

In summary, we developed novel breast tumor-homing yolk-like Gd-Bi NPs (AR-Bi@SiO2-Gd/DOX) as a novel theranostic platform. The NPs could selectively target the MCF-7 tumors, enter the inner part of the tumor tissues and release the drug selectively within the tumors due to the presence of the novel MCF-7 breast tumor-homing peptide (AR). This excellent tumor-homing of the NPs enables the NPs to serve not only as an MRI/CT dual-modal imaging contrast agent for significantly improved tumor detection but also as a synergistic photothermal/chemo-therapeutic agent for significantly effective tumor inhibition. More importantly, the NPs could remain at the tumor sites for at least 14 days, achieving ultralong retention time. They were then cleared from the main organs and excreted from the body along with the faeces and urine. This ultralong retention along with their targeted entry into the inner part of the tumor tissues would enable the proposed NPs to hold promise for an effective deep-tissue tumor imaging-guided therapy.

Experimental

Experimental methods are available in the ESI.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We acknowledge the financial support received from the National Natural Science Foundation of China (51672085, 21620102004, 31700880, 31771038), Postdoctoral Fund (2018M640776), China Postdoctoral Innovation Talent Supporting Program (BX20190150), Presidential Foundation of Zhujiang Hospital (yzjj2018rc09) and Scientific Research Foundation of Southern Medical University (C1051353 and PY2018N060). Y. Z. and C. M. would like to acknowledge the financial support received from the National Institutes of Health (EB021339).

References

  1. B. Yang, Y. Chen and J. Shi, Chem, 2018, 4, 1284–1313 CAS.
  2. M. Liu, Q. Li, L. Liang, J. Li, K. Wang, J. Li, M. Lv, N. Chen, H. Song, J. Lee, J. Shi, L. Wang, R. Lal and C. Fan, Nat. Commun., 2017, 8, 15646 CrossRef CAS PubMed.
  3. X. Liu, H. Jiang, J. Ye, C. Zhao, S. Gao, C. Wu, C. Li, J. Li and X. Wang, Adv. Funct. Mater., 2016, 20, 8694–8706 CrossRef.
  4. S. P. Meyers, Am. J. Roentgenol., 2007, 193, W152 Search PubMed.
  5. D. Choi, S. H. Kim, J. H. Lim, J. M. Cho, W. J. Lee, S. J. Lee and H. K. Lim, J. Comput. Assist. Tomogr., 2001, 25, 777–785 CrossRef CAS PubMed.
  6. S. Gangi, J. G. Fletcher, M. A. Nathan, J. A. Christensen, W. S. Harmsen, B. S. Crownhart and S. T. Chari, Am. J. Roentgenol., 2004, 182, 897–903 CrossRef PubMed.
  7. S. Kobayashi, S. Tsuruoka, Y. Usui, H. Haniu, K. Aoki, S. Takanashi, M. Okamoto, H. Nomura, M. Tanaka, S. Aiso, M. Saito, H. Kato and N. Saito, NPG Asia Mater., 2015, 7, e203 CrossRef CAS.
  8. T. Kim, N. Lee, D. R. Arifin, I. Shats, M. Janowski, P. Walczak, T. Hyeon and B. Jwm, Adv. Funct. Mater., 2017, 27, 1604213 CrossRef PubMed.
  9. C. Xu, Y. Wang, C. Zhang, Y. Jia, Y. Luo and X. Gao, Nanoscale, 2017, 9, 4620 RSC.
  10. J. Liu, X. Zheng, L. Yan, L. Zhou, G. Tian, W. Yin, L. Wang, Y. Liu, Z. Hu and Z. Gu, ACS Nano, 2016, 12, 696–707 Search PubMed.
  11. P. Lei, R. An, P. Zhang, S. Yao, S. Song, L. Dong, X. Xu, K. Du, J. Feng and H. Zhang, Adv. Funct. Mater., 2017, 27, 1702018 CrossRef.
  12. F. Mao, L. Wen, C. Sun, S. Zhang, G. Wang, J. Zeng, Y. Wang, J. Ma, M. Gao and Z. Li, ACS Nano, 2016, 10, 11145 CrossRef CAS PubMed.
  13. K. D. Mjos and C. Orvig, Chem. Rev., 2014, 114, 4540 CrossRef CAS PubMed.
  14. L. Li, Y. Lu, C. Jiang, Y. Zhu, X. Yang, X. Hu, Z. Lin, Y. Zhang, M. Peng, H. Xia and C. Mao, Adv. Funct. Mater., 2018, 28, 1704623 CrossRef PubMed.
  15. A. Detappe, E. Thomas, M. W. Tibbitt, S. Kunjachan, O. Zavidij, N. Parnandi, E. Reznichenko, F. Lux, O. Tillement and R. Berbeco, Nano Lett., 2017, 17, 1733–1740 CrossRef CAS PubMed.
  16. B. Wu, S. T. Lu, H. Yu, R. F. Liao, H. Li, B. V. L. Zafitatsimo, Y. S. Li, Y. Zhang, X. L. Zhu and H. G. Liu, Biomaterials, 2047, 159, 37–47 CrossRef PubMed.
  17. M. Yang, K. Sunderland and C. Mao, Chem. Rev., 2017, 117, 10377–10402 CrossRef CAS PubMed.
  18. X. Huang, F. Zhang, S. Lee, M. Swierczewska, D. O. Kiesewetter, L. Lang, G. Zhang, L. Zhu, H. Gao, H. S. Choi, G. Niu and X. Chen, Biomaterials, 2012, 33, 4370–4378 CrossRef CAS PubMed.
  19. H. He, X. Zheng, J. Zhang, S. Liu, X. Hu and Z. Xie, J. Mater. Chem. B, 2017, 5, 2491–2499 RSC.
  20. Q. Xia, Z. Chen, Z. Yu, L. Wang, J. Qu and R. Liu, ACS Appl. Mater. Interfaces, 2018, 10, 17081–17088 CrossRef CAS PubMed.
  21. J. Lin, X. Zeng, Y. Xiao, L. Tang, J. Nong, Y. Liu, H. Zhou, B. Ding, F. Xu, H. Tong, Z. Deng and X. Hong, Chem. Sci., 2019, 10, 1219–1226 RSC; X. Qu, P. Qiu, Y. Zhu, M. Yang and C. Mao, NPG Asia Mater., 2017, 9, e452 CrossRef CAS PubMed.
  22. X. Qu, P. Qiu, Y. Zhu, M. Yang and C. Mao, NPG Asia Mater., 2017, 9, e452 CrossRef CAS PubMed.
  23. B. D. Anderson, W. C. Wu and J. B. Tracy, Chem. Mater., 2016, 28, 4945–4952 CrossRef CAS.
  24. M. Kanezashi, M. Sano, T. Yoshioka and T. Tsuru, Chem. Commun., 2010, 46, 6171–6173 RSC.
  25. K. Daigoku, A. Okada and K. Nakada, Chem. Phys. Lett., 2006, 430, 221–226 CrossRef CAS.
  26. J. W. Wojtkowiak, D. Verduzco, K. J. Schramm and R. J. Gillies, Mol. Pharmaceutics, 2011, 8, 2032 CrossRef CAS PubMed.
  27. V. P. Torchilin, Nat. Rev. Drug Discovery, 2014, 13, 813 CrossRef CAS PubMed.
  28. L. Chen, L. Li, L. Zhang, S. Xing, T. Wang, Y. A. Wang, C. Wang and Z. Su, ACS Appl. Mater. Interfaces, 2013, 5, 7282 CrossRef CAS PubMed.
  29. M. Shilo, T. Reuveni, M. Motiei and R. Popovtzer, Nanomedicine, 2012, 7, 257–269 CrossRef CAS PubMed.
  30. N. Lee, S. H. Choi and T. Hyeon, Adv. Mater., 2013, 25, 2641–2660 CrossRef CAS PubMed.
  31. V. Torchilin, Adv. Drug Delivery Rev., 2011, 63, 131–135 CrossRef CAS PubMed.
  32. N. Bertrand, J. Wu, X. Xu, N. Kamaly and O. C. Farokhzad, Adv. Drug Delivery Rev., 2014, 66, 2–25 CrossRef CAS PubMed.
  33. J. Wang, J. Liu, Y. Liu, L. Wang, M. Cao, Y. Ji, X. Wu, Y. Xu, B. Bai, Q. Miao, C. Chen and Y. Zhao, Adv. Mater., 2016, 28, 8950–8958 CrossRef CAS PubMed.
  34. D. Jaque, L. Martinez Maestro, B. del Rosal, P. Haro-Gonzalez, A. Benayas, J. L. Plaza, E. Martin Rodriguez and J. Garcia Sole, Nanoscale, 2014, 6, 9494–9530 RSC.
  35. A. Xu, L. Zhang, J. Yuan, F. Babikr, A. Freywald, R. Chibbar, M. Moser, W. Zhang, B. Zhang, Z. Fu and J. Xiang, Cell. Mol. Immunol., 2018 DOI:10.1038/s41423-018-0184-y.
  36. K. F. Chu and D. E. Dupuy, Nat. Rev. Cancer, 2014, 14, 199 CrossRef CAS PubMed.
  37. T. M. Sun, Y. S. Zhang, B. Pang, D. C. Hyun, M. X. Yang and Y. N. Xia, Angew. Chem., Int. Ed., 2014, 53, 12320–12364 CAS.
  38. E. Sulheim, J. Kim, A. van Wamel, E. Kim, S. Snipstad, I. Vidic, I. H. Grimstad, M. Widerøe, S. H. Torp, S. Lundgren, D. J. Waxman and C. de Lange Davies, J. Controlled Release, 2018, 279, 292–305 CrossRef CAS PubMed.
  39. B. Haley and E. Frenkel, Urol. Oncol., 2008, 26, 57–64 CrossRef CAS PubMed.
  40. J. Lu, M. Liong, Z. Li, J. I. Zink and F. Tamanoi, Small, 2010, 6, 1794–1805 CrossRef CAS PubMed.
  41. F. Peng, M. I. Setyawati, J. K. Tee, X. Ding, J. Wang, M. E. Nga, H. K. Ho and D. T. Leong, Nat. Nanotechnol., 2019, 14, 279–286 CrossRef CAS PubMed.
  42. F. Peng, J. K. Tee, M. I. Setyawati, X. Ding, H. L. A. Yeo, Y. L. Tan, D. T. Leong and H. K. Ho, ACS Appl. Mater. Interfaces, 2018, 10, 31938–31946 CrossRef CAS PubMed.
  43. J. Wang, L. Zhang, F. Peng, X. Shi and D. T. Leong, Chem. Mater., 2018, 30, 3759–3767 CrossRef CAS.
  44. M. I. Setyawati, C. Y. Tay, S. L. Chia, S. L. Goh, W. Fang, M. J. Neo, H. C. Chong, S. M. Tan, S. C. J. Loo, K. W. Ng, J. P. Xie, C. N. Ong, N. S. Tan and D. T. Leong, Nat. Commun., 2013, 4, 1673 CrossRef CAS PubMed.

Footnotes

Electronic supplementary information (ESI) available: Experimental methods and detailed results. See DOI: 10.1039/c9mh00014c
These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2019