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A versatile theranostic nanoplatform based on mesoporous silica
Materials Science & Engineering C 98 (2019) 560–571
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
A versatile theranostic nanoplatform based on mesoporous silica a,1
c,1
a
a
Ying Zhang , Jiejun Cheng , Niannian Li , Ruochen Wang , Gang Huang ⁎⁎ Dannong Hea,b,
d,⁎
, Jun Zhu
b,⁎
T
,
a
School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China National Engineering Research Center for Nanotechnology, Shanghai 200241, PR China Department of Radiology, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, PR China d Shanghai University of Medicine and Health Sciences, Shanghai 200093, PR China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Multifunctional theranostic nanoplatform CT imaging MR imaging Chemotherapy
A versatile of mesoporous silica is designed and operated, including ion doping, surface modify and pore adsorption, based on aqueous well-dispersed. Thus, a multifunctional theranostic nanoplatform is obtained through endowing some functional materials. Detailedly, Gd ions is introduced to mesoporous silica (GM nanoparticles) via a co-assemble process, which is used as prime carrier with MRI. Furthermore, the surface graft of hyaluronic acid (HA) molecule makes contribution to lymph system-targeted delivery (GMH nanoparticles). Additionally, the introduction of functional molecules including Iopamidol (IGMH nanoparticles) and DOX (DGMH nanoparticles) could combine the diagnosis and therapy with CT and sustained drug release. We present evidence that the IGMH and DGMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and highlight CT and MR imaging of IGMH nanoparticles in lymph system, and chemotherapy and MR imaging of DGMH nanoparticles in lymph cancer. Our results provide a new universal manufacture for mesoporous silica to obtain a multifunctional theranostic nanoplatform, has great potential for use in biological applications.
1. Introduction The theranostic nanoplatform, a kind of nanocarriers composed by imaging and treatment agents, have realized precise diagnosis and individualized therapeutic strategies [1–4]. A variety of nanocarriers have been developed for nanotheranostics including liposomes, polymer conjugations, inorganic material, and so on [5], in which different imaging modalities, including optical, ultrasound, MR, PET and CT imaging [6], are integrated for maximizing their advantages through the complementary diagnostic information, and some typical therapy, including chemotherapy, gene, photothermal, photodynamics, radiation and immunization therapy, is applied through loading the functional molecules for controlled and targeted release. Furthermore, combining imaging with therapeutic modalities can achieve visualization therapy mediated by imaging for tracking surgical procedure, pharmacokinetic distribution and metabolites [7–11]. Now, many nanomaterials are being developed to carriers for increasing the levels of theranostic agents. Meanwhile, precise design, simple manufacture and good prospect for application are a trend for choosing nanocarriers. Among them, mesoporous material is a popular
and persistent nanocarrier [12]. Specially, more and more functionalized mesoporous silica has been prepared and applied [13,14]. According to the previous papers, we can find some value reasons of wellreceived mesoporous silica as following [15]. Firstly, compared to soft carriers, the preparation of mesoporous silica is a controlled synthesis process, which is simple to obtain the given structure, morphology and pore size. In addition, convenient doping and easy surface modification make the function of mesoporous silica more convenient. However, because mesoporous silica is always easy to be agglomerated in the water, it is still a challenge for the application in some tissues, such as a tiny tumor or its metastatic tumor, intravascular plate, lymph capillaries, and so on. Tumor lymphatic metastasis is one of important content for the major of cancer death. Many researches about theranostics of lymph metastasis tumor have been reported [16–20]. For example, Qiao et al. reported an albumin-based theranostic nano-agent for dual-modal imaging guided photothermal therapy to inhibit lymphatic metastasis of cancer. In our previous experiment, we reported a multifunctional lymph-targeted platform based on Mn@mSiO2 nanocomposites combining PFOB for dual-mode imaging and DOX for diagnose and
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Corresponding authors. Correspondence to: D. He, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China. E-mail addresses: [email protected] (G. Huang), [email protected] (J. Zhu), [email protected] (D. He). 1 These authors contributed equally. ⁎⁎
https://doi.org/10.1016/j.msec.2019.01.004 Received 16 October 2017; Received in revised form 9 August 2018; Accepted 2 January 2019 Available online 03 January 2019 0928-4931/ © 2019 Published by Elsevier B.V.
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Fig. 1. Schematic illustration of the formation of FGMH nanoparticles.
obtained by centrifuging and washing with deionized water three times and then lyophilized.
treatment of lymph tumor [21]. Furthermore, HA molecule was modified to the nanocomposites for in vitro and in vivo targeting of the lymph system. In the present research, our goal is to develop a more effective targeted nanotheranostic based on aqueous well-dispersed mesoporous silica with multifunctional characteristics for CT/MRI imaging, active targeting and chemotherapy applications. Detailedly, gadolinium ions are introduced to mesoporous silica (GM nanoparticles) via a co-assemble process and the surface graft of HA molecule makes contribution to lymph system-targeted delivery (GMH nanoparticles). Additionally, the introduction of functional molecules including Iopamidol (IGMH nanoparticles) and DOX (DGMH nanoparticles) could combine the diagnosis and therapy with CT and sustained drug release. We present evidence that the IGMH and DGMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and highlight MR and CT imaging of IGMH nanoparticles in lymph system, and MR imaging and chemotherapy of DGMH nanoparticles in cancer.
2.3. Preparation of DOX-Gd-mSiO2-HA nanoparticles 0.1 g of GMH nanoparticles was dispersed in 30 mL deionized water, and then 500 μL, 2 mg/mL of doxorubicin (DOX) was added. After being stirred for 24 h, GMH nanoparticles with DOX (DOX-Gd-mSiO2HA, DGMH nanoparticles) were fabricated. DGMH nanoparticles were washed with deionized water to remove the free DOX in the solutions of nanoparticles, until the supernatant liquid was clear. 2.4. FITC labelled GMH nanoparticles 2 mg GMH dispersed in 2 mL 0.02 mg/mL FITC aqueous solution for 24 h at room temperature to avoid light. The modified GMH nanoparticles were washed and centrifuged three times with deionized water and then dried at 60 °C for 12 h.
2. Materials and methods 2.5. Characterization 2.1. Preparation of Gd-mSiO2-HA nanoparticles The morphology of the samples were determined via TEM (JEOL, JEM-2100F, 200 kV) and FESEM (Hitachi, S-4800, 10 kV), respectively. Powder X-ray diffraction (XRD) patterns of samples were recorded on a Rigaku D/MAX-2250V diffractometer using Cu-Ka radiation (40 kV and 40 mA). The FT-IR spectra were collected on a Nicolet 6700 FT-IR spectrometer. Si, Gd, and O in the sample of GMH nanoparticles were further confirmed by 50 mm2 EDX (Oxford). The UV–vis spectra were recorded via PerkinElmer Lambda 950 UV–vis spectrophotometer. The BET surface area was determined via a micromeritics ASAP 2020 instrument. The content of Gd was evaluated by ICP-MS using a PLASMASPEC-II instrument (Horiba Jobin Yvon). The size and zeta potential of nanoparticles was measured via Malvern Zetasizer Nano ZS.
Gadolinium-doped mesoporous silica (Gd-mSiO2, GM) nanoparticles were synthesized as the following: 0.44 g of cetyltrimethyl ammonium bromide (CTAB) was dissolved in 180 mL of deionized water at room temperature. And then 0.24 g of NaOH and 2.08 g of triethoxysilane (TEOS) was added to the solution under rigorous stirring [22]. Furthermore, 0.04 g GdCl3∙6H2O were added rapidly. The precipitates were obtained by filtration after 18 h of stirring. Finally, we got Gd-mSiO2 nanoparticles by washing and refluxing in 7.2 mg/mL ammonium nitrate ethanol solution to remove CTAB remained. The preparation of Gd-mSiO2-HA (GMH) nanoparticles included the surface modification of GM nanoparticles via 3-aminopropyltriethoxysilane (APTES) and the conjugation of hyaluronic acid (HA) on them. In the experiment, 0.2 g of GM nanoparticles was added in 220 μL APTES and 20 mL toluene. The amino-modified GM nanoparticles were washed and centrifuged three times with methanol and deionized water respectively, and then dried at 60 °C for 12 h. Furthermore, HA was activated using N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Subsequently, the obtained amino-modified GM nanoparticles and 2.5 g of HA were added in 400 mL deionized water. Then 0.46 g of EDC and 0.28 g of NHS were dissolved into the solution and stirred at room temperature for 24 h. Finally, the resulting products were obtained by centrifuging and washing with ethanol and deionized water three times respectively, and then drying at 60 °C.
2.6. Cell viability experiments The cells (5 × 103 cells per well) were seeded onto 96-well cell culture plates. HCT116 cells were incubated overnight at 37 °C under 5% CO2. The prepared DGMH nanoparticles and DOX with same concentrations of DOX 0, 4, 8, 12 and 16 mg/mL in PBS were added to the wells respectively for further 4 h. Then, all nanoparticles were removed from the wells. And then 10% CCK solution was added to all wells and incubated for 4 h at 37 °C. The formula was used to calculate the viability of cell growth as follow: Viability (%) = mean of absorbance value of treatment group/mean absorbance value of control × 100%. The results were expressed as an average of five measurements.
2.2. Preparation of Iopamidol-Gd-mSiO2-HA nanoparticles
2.7. MRI in vitro
0.1 g of GMH nanoparticles was dispersed in 30 mL deionized water, and then 2 mL of Iopamidol was added. The mixed solution was kept stirring overnight. The resulting products (IGMH nanoparticles) were
All MRI scans were carried out with a 3.0 T Intera Achieva whole body system (Philips Medical Systems, Best). The DGMH nanoparticles with different mass concentrations were prepared for T1-weighted MRI. 561
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Fig. 2. TEM images of (a) GM and (b) GMH nanoparticles, (c) XRD pattern and (d) N2 adsorption/desorption isotherm of GM and GMH nanoparticles, (e) EDX of GMH nanoparticles and (f) FT-IR spectra of GM and GMH nanoparticles. (g) DLS data of GMH nanoparticles (h) pore size distribution curve of GMH nanoparticles.
2.8. Confocal imaging of cells
The sequence parameters were TR/TE, 4.6/2.4 ms; effective slice thickness, 0.6 mm; matrix, 320 × 256; flip angle, 12°; FOV, 26 cm. The 3D MR image was then reconstructed using maximum-intensity projection (MIP) from the original data set.
The cells were performed using a laser scanning confocal microscopy (Leica, Wetzlar). 1 × 106 cells/mL of HEK293 and HCT116 cells were incubated with DGMH nanoparticles and FITC labeled GM and GMH nanoparticles for 2 h for confocal imaging, fixed with 4% 562
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Fig. 3. (a) T1-weighted images of different Gd concentrations in vitro, (b) the linear relationship between 1/T1 relaxation rates, and T1-weighted MR images in vivo of GMH nanoparticles (right leg) and commercial Gd-DTPA (left leg) injection in rabbit knee popliteal fossa. Scanning time after injection: (c) 15, (d) 30, (e) 45 and (f) 60 min. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
2.11. MRI in vivo in tumor bearing mice
paraformaldehyde for 30 min and stained by DAPI for 8 min. All the cells were washed twice with PBS before confocal imaging. Imaging of DGMH and FITC labeled nanoparticles was carried out laser excitation at 488 nm, and emission from 550 to 570 nm.
7 weeks of nude mouse born were anaesthetized by intraperitoneal injection of 3% pentobarbital solution. The injection site was carefully disinfected. 400 μL GMH nanoparticles (0.10 mg/mL) and the contrast agents were injected intratumor. The interesting MRI area was observed the whole body.
2.9. Immunofluorescence analysis Specific pathogen-free nude mice age 5 weeks, were supplied by Shanghai Jiaotong University. All animal experiments were carried out in accordance with local governmental authorities. The mice carried HCT116 (Human colon cancer cell line) tumor and A549 (Human lung cancer cell line) tumor were established by subcutaneous injection of 1 × 106 tumor cells suspended in 150 μL PBS. And FITC labeled GMH nanoparticles were injected into tumor. The mice were sacrificed to obtain the tumors, after 0.5 h post-injection. These tumors were fixed in 4% paraformaldehyde for 12 h, soaked in 30% sucrose in PBS for 24 h, and then frozen in OCT embedding medium, finally stored in liquid nitrogen. The tumors were fixed in acetone at 4 °C ice-cold for 15 min, washed using PBS and blocked with Bovine Serum Albumin for 1 h. Sections of tumor were incubated with Cy5 labeled rabbit anti-mouse CD44 (2 μg/mL, Shanghai Yu Bo Biotech Co., Ltd.) to label receptors in HCT116 cells for 1 h. By DAPI for nuclear counterstain, the slides of tumor were treated and visualized by fluorescent microscope. Imagings were carried out at 488 nm laser excitation, emission collected from 550 to 570 nm for FITC labeled nanoparticles, but Cy5 at 633 nm laser excitation and emission from 650 to 680 nm.
2.12. Tissue distribution To evaluate the distribution and metabolism of GMH nanoparticles after injected into the body, we use FITC labeled GMH to track the nanoparticles. The short-term metabolism of GMH nanoparticles in vivo was performed as following steps: Firstly, FITC labeled GMH nanoparticles were dissolved in physiological saline at the potency of 2 mg/ mL. Next, 0.1 mL of FITC labeled GMH was injected through the tail vein. The mice were anaesthetized by respiratory anesthesia machine for 1 min. Then the fluorescence distribution of FITC was observed every 0.5 h. After the experiment, all of the mice were sacrificed and about 120 μL of blood sample was obtained firstly. Furthermore, next to blood, brain, lung, liver, heart, kidney and spleen were collected, which were weighed and homogenized. The Gd content was determined by inductively coupled plasma mass spectrometry (ICP-MS), after the process of digesting sample.
2.13. DOX loading and in vitro drug release DGMH nanoparticles 5 mg was dispersed in 10 mL phosphate buffer saline solution (PBS, pH = 5.5), which was introduced into the molecular weight cut-off is 8000–14,000 dialysis cassettes. Furthermore, the dialysis cassettes were placed in PBS (100 mL) as the release medium. 10 mL of release medium was taken out for the determination of UV spectra around 490 nm at given time intervals. The same volume of fresh buffer solution was added. The curve of the release rate for DOX was researched between the cumulative release amount and time t.
2.10. MRI in vivo in rabbit New Zealand White Rabbits with the weights of 3.5 kg were anaesthetized by intraperitoneal injection of 3% pentobarbital solution, whose temperature was kept at physiological level during whole experimental process, 400 μL GMH nanoparticles (0.10 mg/mL) and Gadopentetate were injected into the right and left knee popliteal fossa respectively. The interesting MRI area was observed around the upper thigh. 563
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Fig. 4. In vitro laser scanning confocal microscopy images of HCT116 and HEK293 cells cultivated with GM and GMH nanoparticles.
The target ability of DGMH nanoparticles on HCT116 tumor cells was obtained by CCK assay according to the previously reported procedure. The cells were cultured for 24 h. The dose of DOX from DGMH was added as much as free DOX for two days and removed, and CCK solution was added and incubated for 4 h and removed, and PBS was added to the wells. The cell viability was detected using iMark/xMark Bio-Rad microplate reader at 450 nm.
The tumor mice were randomly divided into two groups with six mice in each group. They were subcutaneously injected with 0.2 mL DGMH (0.10 mg/mL) nanoparticles dispersed in PBS and the same volume PBS respectively when the diameter of tumor was about 10 mm. After 6 days, the tumor mice were treated again the same conditions. The tumor volumes were calculated by (L × W2)/2, where L and W were length and width. All mice were euthanized and the tumors were weighed on the 24th day. At each time point postinjection, the tumor volume, body weight were recorded.
2.15. Growth inhibition of DGMH nanoparticles on tumors in vivo
2.16. Statistical analysis
Mouse lymphoma cells YAC-1 tumor bearing mice were established by subcutaneous injection of 1 × 106 cells suspended in 150 μL PBS.
Results are expressed as the mean ± standard deviation (SD). Statistical analysis was done using the Student's t-test. There was a
2.14. Growth inhibitory effect of DGMH nanoparticles on tumor cell
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Fig. 5. Immunofluorescence images of HCT116 and A549 tumor cultivated with GMH nanospheres. All images were taken under the identical instrumental conditions and presented at the same intensity scale.
Finally, the introduction of functional molecules including Iopamidol and DOX could combine the diagnosis and therapy with CT and sustained drug release. In a word, functional GMH (FGMH) is designed for lymph-targeted diagnosis and therapy. The whole procedure is schematically illustrated in Fig. 1. The morphology and structure of the obtained nanoparticles are characterized. As shown in Fig. 2a. The diameter of GM nanoparticles are uniform with an average diameter of about 150 nm and the typical mesoporous structure of GM nanoparticles with uniform pore opening is observed. Furthermore, GMH nanoparticles are viewed, whose structure has not changed (Fig. 2b). The small angle XRD patterns reveal that main diffraction peak (100) of typical mesoporous silica is obtained, which shows that mesoporous silica is maintained although the possible destroy of the channel frameworks by doping Gd ions. Moreover, GMH nanoparticles still have a low (100) diffraction peak, which implies that the introduction of HA strongly fills the mesoporous channel (Fig. 2c). Additionally, as shown in Fig. 2d, a typical type IV isotherm with a steep capillary condensation step of GM and GMH nanoparticles indicates the characteristic of an excellent quality mesoporous material, whose surface area of GM and GMH nanoparticles is decreased from 527 to 353 m2/g. Specially, the loading of Gd ions and HA molecules are proved by EDX and FT-IR, respectively. EDX reveals that the Gd content is about 3.5% in GMH nanoparticles. Furthermore, ICP-MS reveals that the Gd content is 31.12 mg/g. The typical peaks of HA in GMH nanoparticles, such as 1640 and 1400 cm−1 are observed, which comes from the characteristic vibrations of C]O. The above results indicate that the desired products are prepared successfully. The average size of uniform GMH nanoparticles is 137 nm, and the pore size is about 3 nm (Fig. 2g–h). These indicate that HA molecules have successfully modified mesoporous channels and have not destroyed the mesoporous structure of drug carriers. To evaluate the MRI property of GMH nanoparticles, T1-weighted MR images in vitro and in vivo were obtained. As shown in Fig. 3a, different Gd concentrations of GMH nanoparticles in the centrifuge tubes, as well as deionized water for the background signal, are measured for their T1 relaxation time by a 3 T MR imaging scanner. The result shows that the T1-weighted MRI signal intensity is continuously enhanced, leading to the brighter images with increasing Gd content. Furthermore, as shown in Fig. 3b, the longitudinal proton relaxation
Fig. 6. In vivo bio-distribution of GMH nanoparticles by tail vein administration at the time of (a) controlled group, (b) 0.5, (c) 1, (d) 1.5, (e) 2, (f) 2.5 and (g) 3 h after single injection, and (h) mean Gd concentrations (mg/kg) per gram organ over time after consecutive injection.
statistically significant difference at a P value of less than 0.05.
3. Results and discussion GM nanoparticles are obtained by doping Gd ions to mesoporous silica material via a co-assemble process, which is used as prime carrier with MRI from Gd ions [24]. Furthermore, the surface graft of HA molecule makes contribution to lymph system-targeted delivery. 565
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Fig. 7. (a) TEM image and (b) SEM image, (c) N2 adsorption/desorption isotherm of IGMH nanoparticles and (d) pore size distribution curve of IGMH nanoparticles.
probe, while HEK293 cells are low expression of endogenous ligand receptor as control groups. As shown in Fig. 4, compared to the low interaction of HCT116 cells and GM nanoparticles (Fig. 4a3), HAcoated GMH nanoparticles are embraced into the cells largely and tightly distribute among whole cells (Fig. 4b3). The result reveals that GMH has strong affinity towards HCT116 cells. However, both GM and GMH nanoparticles express poor ability of phagocytosis on HEK293 cells (Fig. 4c3 and d3). These results imply that HA-coated GMH nanoparticles is high targeting efficiency to CD44, which implies that the obtained products is potential in lymph system-targeted because of 43% homology as homologues between CD44 and lymphatic endothelial hyaluronan receptor-1 (LYVE-1). Furthermore, the targeting ability of the GMH nanoparticles to lymph system was estimated by immunofluorescence. The principle of targeting is analyzed between HA and CD44 receptor by immunofluorescence studies. The rabbit anti-mouse CD44 is red recorded with Cy5 filter, tissue cells is blue dyed with DAPI and GMH nanoparticles are labeled in green with a rhodamine green filter. As shown in Fig. 5, HCT116 tumor possesses high expression of CD44 and GMH nanoparticles selectively localizes within HCT116 tumor, which means the colocalization of GMH nanoparticles with lymphatic vessel markers CD44 receptor. However, CD44 are almost invisible in A549 tumor tissue, and the distribution of GMH in A549 tumor tissue with low expression of CD44 is not uniform. These results indicate that GMH nanoparticles could bind to CD44 receptor specifically, which contributes to the targeting ability of HA-based nanoparticles to lymphatic system. In order to evaluate the distribution and metabolism of GMH nanoparticles in body, FITC labeled GMH nanoparticles were used to track their distribution with in vivo imaging. As shown in Fig. 6a–g, 0.1 mL of GMH-FITC nanoparticles is injected by tail vein administration. Some abdominal organs, such as spleen, liver, colon, and so on, are found at the time of 0.5 h (Fig. 6b). With the increasing of injection time to 1 h, GMH-FITC nanoparticles are transferred to the lung, heart, brain
rate as a function of Gd ion concentrations results in an r1 relaxivity of 11.2 mM−1∙s−1, which is much larger than that of Magnevist (r1 = 4.5 mM−1∙s−1). Moreover, the MR imaging ability of the GMH nanoparticles injected in the right lymph node are evaluated in vivo compared to that of commercial Gd-DTPA injected in the left lymph node. As shown in Fig. 3c, the left lymph node (labeled with red arrow) is imaged within 15 min, but the right lymph node is not observed and only some lymph vessels around the lymph node (labeled with white arrow) are visible. The result probably attributes to the different transportation channel, where Gd-DTPA could be transferred from blood vessel and lymph vessel while GMH nanoparticles coating by HA molecule only by lymph vessel, which exhibit the distinct lymph targeting ability of the probe [14]. Increasing the transport time, the signal intensity of left lymph node becomes stronger but that of the right lymph node is still invisible at the time of 30 min (Fig. 3d). However, with the enhancement time of 45 min, the left lymph node is obscure but the right lymph node (labeled with yellow arrow) become clear, suggesting that Gd-DPTA is metabolized while GMH nanoparticles is assembled in the lymph node (Fig. 3e). Moreover, Gd-DPTA is further metabolized, which result in the lower signal of the left lymph node. However, the signal of right lymph node is still strong, which means that GMH nanoparticles provide a longer imaging ability for lymph system. Therefore, these results illustrate that GMH nanoparticles are targeted to lymph system and the enhancement caused by GMH nanoparticles is higher than that of the commercial Gd-DPTA in vitro and in vivo. Based on previous studies, the principal cellular uptake mechanism is the interaction between HA and the CD44 receptor, that is receptormediated endocytosis [25,26]. Cellular uptake efficiency of the GMH nanoparticles was observed by laser scanning confocal microscopy, to evaluate the targeting specificity of the GMH nanoparticles to lymph system. HCT116 and HEK293 cells were dyed with blue fluorescence DAPI and nanoparticles were labeled with green FITC. HCT116 cells are high CD44 receptor-expressing cells which show special affinity to the 566
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Fig. 8. (a) In vitro CT images of IGMH with different concentrations. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
about 200 nm. Moreover, IGMH nanoparticles are viewed, whose porous structure has not changed. Furthermore, as shown in Fig. 2c, a typical type IV isotherm with a steep capillary condensation step of IGMH nanoparticles and the surface area of IGMH nanoparticles is decreased to 211 m2/g. Specially, the pore size is about 3.4 nm (Fig. 7d). These demonstrate that Iopamidol molecules have successfully modified porous channels. CT imaging is a comprehensive and clinically commonly used imaging tools for diagnosis and medical research, in which CT contrast agents are a key factor to enhance imaging. Hence, design and research of multifunctional CT contrast agents is challenging task. In present experiments, Iopamidol was absorbed in GMH nanoparticles to form IGMH nanoparticles with bifunctional diagnosis of MRI and CT imaging. As shown in Fig. 8a, a sharp signal enhancement of CT imaging is found with the increase of IGMH concentrations, which reveals that IGMH nanoparticles are obtained and applied. Furthermore, lymphtargeted CT imaging of the dual-mode contrast agent was performed in vivo in rabbit. An attempt was made to locate lymph nodes in the knee
(Fig. 6c). Furthermore, the GMH-FITC is distributed to various organs by the blood circulation within 1.5 h (Fig. 6d). However, GMH-FITC nanoparticles focus on the kidney and brain at 2 h (Fig. 6e). With further enhancement of injection time, the content of GMH-FITC nanoparticles in the kidney and brain decreases gradually (Fig. 6f), and finally few GMH-FITC nanoparticles are remained in the kidney (Fig. 6g), which indicates that the GMH nanoparticles are likely to be cleared through the kidney. Furthermore, the result of consecutive administration monitored by ICP resulted in accumulation in liver, kidneys and spleen, meaning that these organs may be potential target organs for the of GMH nanoparticles [27], which indicate that the GMH nanoparticles could be conveyed organs through the bloodstream and finally metabolized through liver, kidneys and spleen. In order to evaluate further potential application of the material in tumor imaging, the Iopamidol is loaded on GMH nanoparticles to study the multimode imaging of IGMH nanoparticles. In Fig. 7, the morphology and structure of IGMH nanoparticles are characterized. As shown in Fig. 7a–b. The average diameter of IGMH nanoparticles is 567
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Fig. 9. T1-weighted MRI in vivo of the tumor-bearing nude mice by GMH nanoparticles at different scanning time: (a) 0, (b) 5, (c) 10, (d) 20, (e) 30 and (f) 40 min.
a quite uniform distribution of the nanoparticles within the tumor region, allowing for effective and long-periodic MR imaging of the whole tumor. In order to evaluate further potential application of the material in tumor therapy, the hydrophilic DOX is loaded on GMH nanoparticles to study the therapy function of DGMH nanoparticles. Some material characterizations are obtained and shown in Fig. 10. The surface area of DGMH nanoparticles is 213 m2/g after the mesoporous pores are filled by DOX molecules (Fig. 10b). The UV–vis absorption spectra of DOX at 0.1 mg/mL concentration of the DGMH nanoparticles and DOX are shown (Fig. 10c). The absorption spectra of DOX loading in the DGMH nanoparticles reveals characteristic absorption peaks at 490 nm, while GMH nanoparticles in UV exhibits no obvious absorption peak [28]. The drug loading of nanoparticles were 58.6% using the method of UV spectrophotometry. In order to test the drug loading properties of the materials, the release of DOX of DGMH was shown in Fig. 10d in PBS with acidic conditions (pH = 5.5) in vitro [29]. The final release amount is observed to be 30.1% at 80 h, which illustrates the substantial release of the DOX in vitro. To examine the feasibility of the obtained DGMH in biomedical applications, the targeting specificity and cytotoxicity are investigated. As is shown in Fig. 11, the cellular uptake efficiency of nanoparticles is observed by confocal laser scanning microscopy due to the red light of DOX under 488 nm exciting. It is obviously that DGMH nanoparticles are better surrounded by HCT116 cells, which is abundant expression of CD44 receptor, but few nanoparticles gathering around HEK293 cells. The cell cytotoxicity is examined to evaluate the feasibility and target ability of the DGMH nanoparticles for in vitro growth inhibitory on tumor cells. As shown in Fig. 12a and b, growth inhibition of cells is observed after incubation with free DOX and DGMH nanoparticles. The results indicate the dose-dependent cytotoxicity behavior, which reveals that the DGMH nanoparticles show remarkably higher anticancer
popliteal fossa regions both using IGMH nanoparticles (left leg) and commercial Iopamidol (right leg), which is helpful for observing the structure of the lymph nodes after IGMH nanoparticles administration. When the time is 15 min (Fig. 8b), the lymph signal intensity of injection Iopamidol (labeled with red arrow) is higher than that of IGMH nanoparticles (labeled with yellow arrow). When the time is increased from 30 to 45 min, the lymph vessels are still clear on the left and become invisible on the right (Fig. 8c–d). Furthermore, the image in Fig. 8d is reconstructed three- dimensionally to observe the content of residual contrast agents. As shown in Fig. 8e, IGMH nanoparticles in right leg (labeled with yellow arrow) is higher than that of the commercial Iopamidol in left leg (labeled with red arrow), which demonstrates that the Iopamidol inside the channels of IGMH could be released slowly. Thus, IGMH nanoparticles are a useful dual-mode contrast agent for both MR and CT imaging. In vivo CT images of IGMH nanoparticles (left leg) and commercial Iopamidol (right leg) injection in rabbit knee popliteal fossa. Scanning time after hypodermic injection: (b) 15, (c) 30, (d) 45 min, and (e) 3D Reconstruction on work-station using software. YAC-1 tumor-bearing nude mice were imaging by MR in vivo by intratumorally injection with GMH nanoparticles, which are the excellent MR imaging in vitro and targeting specificity to lymph node. That was evaluated the potential to use GMH nanoparticles for diagnosis of a xenografted tumor model. Compared to the MR image before injection (Fig. 9a), we can clearly see that the typical T1-weighted MR signal of tumor region increases obviously with injection time (Fig. 9b). With the strengthening, some subtle structures in tumor region are observed more distinctly and clearly (Fig. 9c). Moreover, the brightness of tumor region hardly decreases at 20 and 40 min post-injection, which reveals the long residence time in tumor due to the specific binding between GMH nanoparticles and their receptor (Fig. 9d–f). The results suggest that the intra-tumor injection of the GMH nanoparticles leads to 568
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Fig. 10. (a) SEM image and (b) N2 adsorption/desorption isotherm of DGMH nanoparticles, (c) UV–vis absorption spectra of DOX and DGMH nanoparticles and (d) DOX release curve from DGMH nanoparticles.
Fig. 11. Laser scanning confocal microscopy images of HCT116 and HEK293 cells incubated with DGMH nanoparticles.
efficiency on HCT116 cells than that of free DOX. On the contrary, this phenomenon could not be observed in the CCK-8 essays on HEK293 cells (Fig. 12b). We further explored the anti-tumor efficacy of the
DGMH nanoparticles in YAC-1 cell-bearing nude mice in vivo. Twelve of nude mice with YAC-1 tumor cells are randomly divided into two groups (6 rats in each group), administrated with PBS and DGMH 569
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Fig. 12. The inhibitory effect of free DOX and DGMH nanoparticles on (a) HCT116 and (b) HEK293 cells was examined by CCK-8 assay.
Fig. 13. (a) Mean tumor volume of the mice in different groups after treatment, (b) mean tumor weights of the mice in different groups after treatment, (c) photograph of the tumors from different groups after treatment. Data represent the mean standard deviation of six mice.
4. Conclusions
respectively. After 2 weeks feeding, the two groups of nude mice were intratumorally injected with 200 μL DGMH (0.1 mg/mL) nanoparticles and the same volume PBS respectively twice every week. In Fig. 13a, it is obviously that the size of the tumor for the PBS group increased significantly during the experiment. In contrast, the groups injected with the DGMH nanoparticles greatly inhibit the tumor growth, indicating the experiment. In contrast, the groups injected with the DGMH nanoparticles greatly inhibit the tumor growth, indicating the obvious anti-tumor efficacy [30,31]. After 24 days of treatment, all of the tumor-bearing nude mice were sacrificed and their tumors were taken out and weighed, the results shown in Fig. 13b indicate that the mean tumor weight of DGMH group is much lower than that of the control group. And the photograph of the tumors from different groups after treatment are also confirmed this point, (Fig. 13c). The tumor sizes of the PBS group are obviously observed bigger than that of DGMH nanoparticles group. All of the above results reveal that the DMMH nanoparticles are a powerful carrier candidate for combined diagnosis and therapy of cancer in vivo.
In summary, GMH nanoparticles are prepared by doping Gd ions to mesoporous silica material via a co-assemble process and grafting HA molecule on their surface. Furthermore, FGMH nanoparticles are obtained through the introduction of functional molecules including Iopamidol and DOX. Therefore, the active targeted nanotheranostic probe based on FGMH nanoparticles are successfully prepared and used not only as a MRI/CT contrast agent but also as an anti-tumor drug carrier. Moreover, compared with conventional contrast agent, the obtained products have potential challenge to be used as a MRI contrast due to its outstanding T1-weighted MRI signal in vivo. Specially, FGMH nanoparticles also indicate potential application in drug delivery, which presents an excellent performance in inhibiting growth of mice lymphatic tumor. Furthermore, FGMH nanoparticles are potential in high lymph targeting efficiency. Among them, IGMH nanoparticles are a dual-mode contrast agent about both CT and MR imaging for lymph system and DGMH nanoparticles are powerful agents for combined diagnosis and therapy of cancer in vivo. According to our results in the 570
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experiment, not only a new method for uniform nanoparticles but also novel and versatile FGMH nanoparticles are proposed, which could be used as an ideal candidate for simultaneous bio-imaging and chemotherapy for lymphatic system.
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Notes The authors declare no competing financial interest. Acknowledgements This work was supported by National Key R&D Program of China (2016YFA0201200), Shanghai Science and Technology Development Funds (no. 17XD1421900 and 14411968100) and Seed Fund of Renji Hospital of Shanghai Jiao Tong University School of Medicine (no. RJZZ16-010). References [1] S. Mura, P. Couvreur, Nanotheranostics for personalized medicine, Adv. Drug Deliv. Rev. 64 (2012) 1394–1416. [2] L.S. Wang, M.C. Chuang, J.A. Ho, Int. J. Nanomedicine 7 (2012) 4679–4695. [3] M.S. Muthu, D.T. Leong, L. Mei, S.S. Feng, Theranostics 4 (2014) 660–677. [4] Y. Ma, J. Huang, S. Song, H. Chen, Z. Zhang, Small 12 (2016) 4936–4954. [5] L.Y. Rizzo, B. Theek, G. Storm, F. Kiessling, T. Lammers, Curr. Opin. Biotechnol. 24 (2013) 1159–1166. [6] R. Toy, L. Bauer, C. Hoimes, K.B. Ghaghada, E. Karathanasis, Adv. Drug Deliv. Rev. 76 (2014) 79–97. [7] K.M. Rosenblatt, H. Bunjes, Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V (2017), https://doi.org/10.1016/j.ejpb. 2017.03.010. [8] O.S. Muddineti, B. Ghosh, S. Biswas, Expert Opin. Drug Deliv. 14 (2017) 715–726. [9] Y. Han, Y. Li, P. Zhang, J. Sun, X. Li, X. Sun, F. Kong, Pharm. Dev. Technol. 21 (2015) 277–281. [10] J.H. Lee, Y. Yeo, Chem. Eng. Sci. 125 (2015) 75–84.
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Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
A versatile theranostic nanoplatform based on mesoporous silica a,1
c,1
a
a
Ying Zhang , Jiejun Cheng , Niannian Li , Ruochen Wang , Gang Huang ⁎⁎ Dannong Hea,b,
d,⁎
, Jun Zhu
b,⁎
T
,
a
School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China National Engineering Research Center for Nanotechnology, Shanghai 200241, PR China Department of Radiology, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, PR China d Shanghai University of Medicine and Health Sciences, Shanghai 200093, PR China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Multifunctional theranostic nanoplatform CT imaging MR imaging Chemotherapy
A versatile of mesoporous silica is designed and operated, including ion doping, surface modify and pore adsorption, based on aqueous well-dispersed. Thus, a multifunctional theranostic nanoplatform is obtained through endowing some functional materials. Detailedly, Gd ions is introduced to mesoporous silica (GM nanoparticles) via a co-assemble process, which is used as prime carrier with MRI. Furthermore, the surface graft of hyaluronic acid (HA) molecule makes contribution to lymph system-targeted delivery (GMH nanoparticles). Additionally, the introduction of functional molecules including Iopamidol (IGMH nanoparticles) and DOX (DGMH nanoparticles) could combine the diagnosis and therapy with CT and sustained drug release. We present evidence that the IGMH and DGMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and highlight CT and MR imaging of IGMH nanoparticles in lymph system, and chemotherapy and MR imaging of DGMH nanoparticles in lymph cancer. Our results provide a new universal manufacture for mesoporous silica to obtain a multifunctional theranostic nanoplatform, has great potential for use in biological applications.
1. Introduction The theranostic nanoplatform, a kind of nanocarriers composed by imaging and treatment agents, have realized precise diagnosis and individualized therapeutic strategies [1–4]. A variety of nanocarriers have been developed for nanotheranostics including liposomes, polymer conjugations, inorganic material, and so on [5], in which different imaging modalities, including optical, ultrasound, MR, PET and CT imaging [6], are integrated for maximizing their advantages through the complementary diagnostic information, and some typical therapy, including chemotherapy, gene, photothermal, photodynamics, radiation and immunization therapy, is applied through loading the functional molecules for controlled and targeted release. Furthermore, combining imaging with therapeutic modalities can achieve visualization therapy mediated by imaging for tracking surgical procedure, pharmacokinetic distribution and metabolites [7–11]. Now, many nanomaterials are being developed to carriers for increasing the levels of theranostic agents. Meanwhile, precise design, simple manufacture and good prospect for application are a trend for choosing nanocarriers. Among them, mesoporous material is a popular
and persistent nanocarrier [12]. Specially, more and more functionalized mesoporous silica has been prepared and applied [13,14]. According to the previous papers, we can find some value reasons of wellreceived mesoporous silica as following [15]. Firstly, compared to soft carriers, the preparation of mesoporous silica is a controlled synthesis process, which is simple to obtain the given structure, morphology and pore size. In addition, convenient doping and easy surface modification make the function of mesoporous silica more convenient. However, because mesoporous silica is always easy to be agglomerated in the water, it is still a challenge for the application in some tissues, such as a tiny tumor or its metastatic tumor, intravascular plate, lymph capillaries, and so on. Tumor lymphatic metastasis is one of important content for the major of cancer death. Many researches about theranostics of lymph metastasis tumor have been reported [16–20]. For example, Qiao et al. reported an albumin-based theranostic nano-agent for dual-modal imaging guided photothermal therapy to inhibit lymphatic metastasis of cancer. In our previous experiment, we reported a multifunctional lymph-targeted platform based on Mn@mSiO2 nanocomposites combining PFOB for dual-mode imaging and DOX for diagnose and
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Corresponding authors. Correspondence to: D. He, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China. E-mail addresses: [email protected] (G. Huang), [email protected] (J. Zhu), [email protected] (D. He). 1 These authors contributed equally. ⁎⁎
https://doi.org/10.1016/j.msec.2019.01.004 Received 16 October 2017; Received in revised form 9 August 2018; Accepted 2 January 2019 Available online 03 January 2019 0928-4931/ © 2019 Published by Elsevier B.V.
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Fig. 1. Schematic illustration of the formation of FGMH nanoparticles.
obtained by centrifuging and washing with deionized water three times and then lyophilized.
treatment of lymph tumor [21]. Furthermore, HA molecule was modified to the nanocomposites for in vitro and in vivo targeting of the lymph system. In the present research, our goal is to develop a more effective targeted nanotheranostic based on aqueous well-dispersed mesoporous silica with multifunctional characteristics for CT/MRI imaging, active targeting and chemotherapy applications. Detailedly, gadolinium ions are introduced to mesoporous silica (GM nanoparticles) via a co-assemble process and the surface graft of HA molecule makes contribution to lymph system-targeted delivery (GMH nanoparticles). Additionally, the introduction of functional molecules including Iopamidol (IGMH nanoparticles) and DOX (DGMH nanoparticles) could combine the diagnosis and therapy with CT and sustained drug release. We present evidence that the IGMH and DGMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and highlight MR and CT imaging of IGMH nanoparticles in lymph system, and MR imaging and chemotherapy of DGMH nanoparticles in cancer.
2.3. Preparation of DOX-Gd-mSiO2-HA nanoparticles 0.1 g of GMH nanoparticles was dispersed in 30 mL deionized water, and then 500 μL, 2 mg/mL of doxorubicin (DOX) was added. After being stirred for 24 h, GMH nanoparticles with DOX (DOX-Gd-mSiO2HA, DGMH nanoparticles) were fabricated. DGMH nanoparticles were washed with deionized water to remove the free DOX in the solutions of nanoparticles, until the supernatant liquid was clear. 2.4. FITC labelled GMH nanoparticles 2 mg GMH dispersed in 2 mL 0.02 mg/mL FITC aqueous solution for 24 h at room temperature to avoid light. The modified GMH nanoparticles were washed and centrifuged three times with deionized water and then dried at 60 °C for 12 h.
2. Materials and methods 2.5. Characterization 2.1. Preparation of Gd-mSiO2-HA nanoparticles The morphology of the samples were determined via TEM (JEOL, JEM-2100F, 200 kV) and FESEM (Hitachi, S-4800, 10 kV), respectively. Powder X-ray diffraction (XRD) patterns of samples were recorded on a Rigaku D/MAX-2250V diffractometer using Cu-Ka radiation (40 kV and 40 mA). The FT-IR spectra were collected on a Nicolet 6700 FT-IR spectrometer. Si, Gd, and O in the sample of GMH nanoparticles were further confirmed by 50 mm2 EDX (Oxford). The UV–vis spectra were recorded via PerkinElmer Lambda 950 UV–vis spectrophotometer. The BET surface area was determined via a micromeritics ASAP 2020 instrument. The content of Gd was evaluated by ICP-MS using a PLASMASPEC-II instrument (Horiba Jobin Yvon). The size and zeta potential of nanoparticles was measured via Malvern Zetasizer Nano ZS.
Gadolinium-doped mesoporous silica (Gd-mSiO2, GM) nanoparticles were synthesized as the following: 0.44 g of cetyltrimethyl ammonium bromide (CTAB) was dissolved in 180 mL of deionized water at room temperature. And then 0.24 g of NaOH and 2.08 g of triethoxysilane (TEOS) was added to the solution under rigorous stirring [22]. Furthermore, 0.04 g GdCl3∙6H2O were added rapidly. The precipitates were obtained by filtration after 18 h of stirring. Finally, we got Gd-mSiO2 nanoparticles by washing and refluxing in 7.2 mg/mL ammonium nitrate ethanol solution to remove CTAB remained. The preparation of Gd-mSiO2-HA (GMH) nanoparticles included the surface modification of GM nanoparticles via 3-aminopropyltriethoxysilane (APTES) and the conjugation of hyaluronic acid (HA) on them. In the experiment, 0.2 g of GM nanoparticles was added in 220 μL APTES and 20 mL toluene. The amino-modified GM nanoparticles were washed and centrifuged three times with methanol and deionized water respectively, and then dried at 60 °C for 12 h. Furthermore, HA was activated using N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Subsequently, the obtained amino-modified GM nanoparticles and 2.5 g of HA were added in 400 mL deionized water. Then 0.46 g of EDC and 0.28 g of NHS were dissolved into the solution and stirred at room temperature for 24 h. Finally, the resulting products were obtained by centrifuging and washing with ethanol and deionized water three times respectively, and then drying at 60 °C.
2.6. Cell viability experiments The cells (5 × 103 cells per well) were seeded onto 96-well cell culture plates. HCT116 cells were incubated overnight at 37 °C under 5% CO2. The prepared DGMH nanoparticles and DOX with same concentrations of DOX 0, 4, 8, 12 and 16 mg/mL in PBS were added to the wells respectively for further 4 h. Then, all nanoparticles were removed from the wells. And then 10% CCK solution was added to all wells and incubated for 4 h at 37 °C. The formula was used to calculate the viability of cell growth as follow: Viability (%) = mean of absorbance value of treatment group/mean absorbance value of control × 100%. The results were expressed as an average of five measurements.
2.2. Preparation of Iopamidol-Gd-mSiO2-HA nanoparticles
2.7. MRI in vitro
0.1 g of GMH nanoparticles was dispersed in 30 mL deionized water, and then 2 mL of Iopamidol was added. The mixed solution was kept stirring overnight. The resulting products (IGMH nanoparticles) were
All MRI scans were carried out with a 3.0 T Intera Achieva whole body system (Philips Medical Systems, Best). The DGMH nanoparticles with different mass concentrations were prepared for T1-weighted MRI. 561
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Fig. 2. TEM images of (a) GM and (b) GMH nanoparticles, (c) XRD pattern and (d) N2 adsorption/desorption isotherm of GM and GMH nanoparticles, (e) EDX of GMH nanoparticles and (f) FT-IR spectra of GM and GMH nanoparticles. (g) DLS data of GMH nanoparticles (h) pore size distribution curve of GMH nanoparticles.
2.8. Confocal imaging of cells
The sequence parameters were TR/TE, 4.6/2.4 ms; effective slice thickness, 0.6 mm; matrix, 320 × 256; flip angle, 12°; FOV, 26 cm. The 3D MR image was then reconstructed using maximum-intensity projection (MIP) from the original data set.
The cells were performed using a laser scanning confocal microscopy (Leica, Wetzlar). 1 × 106 cells/mL of HEK293 and HCT116 cells were incubated with DGMH nanoparticles and FITC labeled GM and GMH nanoparticles for 2 h for confocal imaging, fixed with 4% 562
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Fig. 3. (a) T1-weighted images of different Gd concentrations in vitro, (b) the linear relationship between 1/T1 relaxation rates, and T1-weighted MR images in vivo of GMH nanoparticles (right leg) and commercial Gd-DTPA (left leg) injection in rabbit knee popliteal fossa. Scanning time after injection: (c) 15, (d) 30, (e) 45 and (f) 60 min. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
2.11. MRI in vivo in tumor bearing mice
paraformaldehyde for 30 min and stained by DAPI for 8 min. All the cells were washed twice with PBS before confocal imaging. Imaging of DGMH and FITC labeled nanoparticles was carried out laser excitation at 488 nm, and emission from 550 to 570 nm.
7 weeks of nude mouse born were anaesthetized by intraperitoneal injection of 3% pentobarbital solution. The injection site was carefully disinfected. 400 μL GMH nanoparticles (0.10 mg/mL) and the contrast agents were injected intratumor. The interesting MRI area was observed the whole body.
2.9. Immunofluorescence analysis Specific pathogen-free nude mice age 5 weeks, were supplied by Shanghai Jiaotong University. All animal experiments were carried out in accordance with local governmental authorities. The mice carried HCT116 (Human colon cancer cell line) tumor and A549 (Human lung cancer cell line) tumor were established by subcutaneous injection of 1 × 106 tumor cells suspended in 150 μL PBS. And FITC labeled GMH nanoparticles were injected into tumor. The mice were sacrificed to obtain the tumors, after 0.5 h post-injection. These tumors were fixed in 4% paraformaldehyde for 12 h, soaked in 30% sucrose in PBS for 24 h, and then frozen in OCT embedding medium, finally stored in liquid nitrogen. The tumors were fixed in acetone at 4 °C ice-cold for 15 min, washed using PBS and blocked with Bovine Serum Albumin for 1 h. Sections of tumor were incubated with Cy5 labeled rabbit anti-mouse CD44 (2 μg/mL, Shanghai Yu Bo Biotech Co., Ltd.) to label receptors in HCT116 cells for 1 h. By DAPI for nuclear counterstain, the slides of tumor were treated and visualized by fluorescent microscope. Imagings were carried out at 488 nm laser excitation, emission collected from 550 to 570 nm for FITC labeled nanoparticles, but Cy5 at 633 nm laser excitation and emission from 650 to 680 nm.
2.12. Tissue distribution To evaluate the distribution and metabolism of GMH nanoparticles after injected into the body, we use FITC labeled GMH to track the nanoparticles. The short-term metabolism of GMH nanoparticles in vivo was performed as following steps: Firstly, FITC labeled GMH nanoparticles were dissolved in physiological saline at the potency of 2 mg/ mL. Next, 0.1 mL of FITC labeled GMH was injected through the tail vein. The mice were anaesthetized by respiratory anesthesia machine for 1 min. Then the fluorescence distribution of FITC was observed every 0.5 h. After the experiment, all of the mice were sacrificed and about 120 μL of blood sample was obtained firstly. Furthermore, next to blood, brain, lung, liver, heart, kidney and spleen were collected, which were weighed and homogenized. The Gd content was determined by inductively coupled plasma mass spectrometry (ICP-MS), after the process of digesting sample.
2.13. DOX loading and in vitro drug release DGMH nanoparticles 5 mg was dispersed in 10 mL phosphate buffer saline solution (PBS, pH = 5.5), which was introduced into the molecular weight cut-off is 8000–14,000 dialysis cassettes. Furthermore, the dialysis cassettes were placed in PBS (100 mL) as the release medium. 10 mL of release medium was taken out for the determination of UV spectra around 490 nm at given time intervals. The same volume of fresh buffer solution was added. The curve of the release rate for DOX was researched between the cumulative release amount and time t.
2.10. MRI in vivo in rabbit New Zealand White Rabbits with the weights of 3.5 kg were anaesthetized by intraperitoneal injection of 3% pentobarbital solution, whose temperature was kept at physiological level during whole experimental process, 400 μL GMH nanoparticles (0.10 mg/mL) and Gadopentetate were injected into the right and left knee popliteal fossa respectively. The interesting MRI area was observed around the upper thigh. 563
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Fig. 4. In vitro laser scanning confocal microscopy images of HCT116 and HEK293 cells cultivated with GM and GMH nanoparticles.
The target ability of DGMH nanoparticles on HCT116 tumor cells was obtained by CCK assay according to the previously reported procedure. The cells were cultured for 24 h. The dose of DOX from DGMH was added as much as free DOX for two days and removed, and CCK solution was added and incubated for 4 h and removed, and PBS was added to the wells. The cell viability was detected using iMark/xMark Bio-Rad microplate reader at 450 nm.
The tumor mice were randomly divided into two groups with six mice in each group. They were subcutaneously injected with 0.2 mL DGMH (0.10 mg/mL) nanoparticles dispersed in PBS and the same volume PBS respectively when the diameter of tumor was about 10 mm. After 6 days, the tumor mice were treated again the same conditions. The tumor volumes were calculated by (L × W2)/2, where L and W were length and width. All mice were euthanized and the tumors were weighed on the 24th day. At each time point postinjection, the tumor volume, body weight were recorded.
2.15. Growth inhibition of DGMH nanoparticles on tumors in vivo
2.16. Statistical analysis
Mouse lymphoma cells YAC-1 tumor bearing mice were established by subcutaneous injection of 1 × 106 cells suspended in 150 μL PBS.
Results are expressed as the mean ± standard deviation (SD). Statistical analysis was done using the Student's t-test. There was a
2.14. Growth inhibitory effect of DGMH nanoparticles on tumor cell
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Fig. 5. Immunofluorescence images of HCT116 and A549 tumor cultivated with GMH nanospheres. All images were taken under the identical instrumental conditions and presented at the same intensity scale.
Finally, the introduction of functional molecules including Iopamidol and DOX could combine the diagnosis and therapy with CT and sustained drug release. In a word, functional GMH (FGMH) is designed for lymph-targeted diagnosis and therapy. The whole procedure is schematically illustrated in Fig. 1. The morphology and structure of the obtained nanoparticles are characterized. As shown in Fig. 2a. The diameter of GM nanoparticles are uniform with an average diameter of about 150 nm and the typical mesoporous structure of GM nanoparticles with uniform pore opening is observed. Furthermore, GMH nanoparticles are viewed, whose structure has not changed (Fig. 2b). The small angle XRD patterns reveal that main diffraction peak (100) of typical mesoporous silica is obtained, which shows that mesoporous silica is maintained although the possible destroy of the channel frameworks by doping Gd ions. Moreover, GMH nanoparticles still have a low (100) diffraction peak, which implies that the introduction of HA strongly fills the mesoporous channel (Fig. 2c). Additionally, as shown in Fig. 2d, a typical type IV isotherm with a steep capillary condensation step of GM and GMH nanoparticles indicates the characteristic of an excellent quality mesoporous material, whose surface area of GM and GMH nanoparticles is decreased from 527 to 353 m2/g. Specially, the loading of Gd ions and HA molecules are proved by EDX and FT-IR, respectively. EDX reveals that the Gd content is about 3.5% in GMH nanoparticles. Furthermore, ICP-MS reveals that the Gd content is 31.12 mg/g. The typical peaks of HA in GMH nanoparticles, such as 1640 and 1400 cm−1 are observed, which comes from the characteristic vibrations of C]O. The above results indicate that the desired products are prepared successfully. The average size of uniform GMH nanoparticles is 137 nm, and the pore size is about 3 nm (Fig. 2g–h). These indicate that HA molecules have successfully modified mesoporous channels and have not destroyed the mesoporous structure of drug carriers. To evaluate the MRI property of GMH nanoparticles, T1-weighted MR images in vitro and in vivo were obtained. As shown in Fig. 3a, different Gd concentrations of GMH nanoparticles in the centrifuge tubes, as well as deionized water for the background signal, are measured for their T1 relaxation time by a 3 T MR imaging scanner. The result shows that the T1-weighted MRI signal intensity is continuously enhanced, leading to the brighter images with increasing Gd content. Furthermore, as shown in Fig. 3b, the longitudinal proton relaxation
Fig. 6. In vivo bio-distribution of GMH nanoparticles by tail vein administration at the time of (a) controlled group, (b) 0.5, (c) 1, (d) 1.5, (e) 2, (f) 2.5 and (g) 3 h after single injection, and (h) mean Gd concentrations (mg/kg) per gram organ over time after consecutive injection.
statistically significant difference at a P value of less than 0.05.
3. Results and discussion GM nanoparticles are obtained by doping Gd ions to mesoporous silica material via a co-assemble process, which is used as prime carrier with MRI from Gd ions [24]. Furthermore, the surface graft of HA molecule makes contribution to lymph system-targeted delivery. 565
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Fig. 7. (a) TEM image and (b) SEM image, (c) N2 adsorption/desorption isotherm of IGMH nanoparticles and (d) pore size distribution curve of IGMH nanoparticles.
probe, while HEK293 cells are low expression of endogenous ligand receptor as control groups. As shown in Fig. 4, compared to the low interaction of HCT116 cells and GM nanoparticles (Fig. 4a3), HAcoated GMH nanoparticles are embraced into the cells largely and tightly distribute among whole cells (Fig. 4b3). The result reveals that GMH has strong affinity towards HCT116 cells. However, both GM and GMH nanoparticles express poor ability of phagocytosis on HEK293 cells (Fig. 4c3 and d3). These results imply that HA-coated GMH nanoparticles is high targeting efficiency to CD44, which implies that the obtained products is potential in lymph system-targeted because of 43% homology as homologues between CD44 and lymphatic endothelial hyaluronan receptor-1 (LYVE-1). Furthermore, the targeting ability of the GMH nanoparticles to lymph system was estimated by immunofluorescence. The principle of targeting is analyzed between HA and CD44 receptor by immunofluorescence studies. The rabbit anti-mouse CD44 is red recorded with Cy5 filter, tissue cells is blue dyed with DAPI and GMH nanoparticles are labeled in green with a rhodamine green filter. As shown in Fig. 5, HCT116 tumor possesses high expression of CD44 and GMH nanoparticles selectively localizes within HCT116 tumor, which means the colocalization of GMH nanoparticles with lymphatic vessel markers CD44 receptor. However, CD44 are almost invisible in A549 tumor tissue, and the distribution of GMH in A549 tumor tissue with low expression of CD44 is not uniform. These results indicate that GMH nanoparticles could bind to CD44 receptor specifically, which contributes to the targeting ability of HA-based nanoparticles to lymphatic system. In order to evaluate the distribution and metabolism of GMH nanoparticles in body, FITC labeled GMH nanoparticles were used to track their distribution with in vivo imaging. As shown in Fig. 6a–g, 0.1 mL of GMH-FITC nanoparticles is injected by tail vein administration. Some abdominal organs, such as spleen, liver, colon, and so on, are found at the time of 0.5 h (Fig. 6b). With the increasing of injection time to 1 h, GMH-FITC nanoparticles are transferred to the lung, heart, brain
rate as a function of Gd ion concentrations results in an r1 relaxivity of 11.2 mM−1∙s−1, which is much larger than that of Magnevist (r1 = 4.5 mM−1∙s−1). Moreover, the MR imaging ability of the GMH nanoparticles injected in the right lymph node are evaluated in vivo compared to that of commercial Gd-DTPA injected in the left lymph node. As shown in Fig. 3c, the left lymph node (labeled with red arrow) is imaged within 15 min, but the right lymph node is not observed and only some lymph vessels around the lymph node (labeled with white arrow) are visible. The result probably attributes to the different transportation channel, where Gd-DTPA could be transferred from blood vessel and lymph vessel while GMH nanoparticles coating by HA molecule only by lymph vessel, which exhibit the distinct lymph targeting ability of the probe [14]. Increasing the transport time, the signal intensity of left lymph node becomes stronger but that of the right lymph node is still invisible at the time of 30 min (Fig. 3d). However, with the enhancement time of 45 min, the left lymph node is obscure but the right lymph node (labeled with yellow arrow) become clear, suggesting that Gd-DPTA is metabolized while GMH nanoparticles is assembled in the lymph node (Fig. 3e). Moreover, Gd-DPTA is further metabolized, which result in the lower signal of the left lymph node. However, the signal of right lymph node is still strong, which means that GMH nanoparticles provide a longer imaging ability for lymph system. Therefore, these results illustrate that GMH nanoparticles are targeted to lymph system and the enhancement caused by GMH nanoparticles is higher than that of the commercial Gd-DPTA in vitro and in vivo. Based on previous studies, the principal cellular uptake mechanism is the interaction between HA and the CD44 receptor, that is receptormediated endocytosis [25,26]. Cellular uptake efficiency of the GMH nanoparticles was observed by laser scanning confocal microscopy, to evaluate the targeting specificity of the GMH nanoparticles to lymph system. HCT116 and HEK293 cells were dyed with blue fluorescence DAPI and nanoparticles were labeled with green FITC. HCT116 cells are high CD44 receptor-expressing cells which show special affinity to the 566
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Fig. 8. (a) In vitro CT images of IGMH with different concentrations. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
about 200 nm. Moreover, IGMH nanoparticles are viewed, whose porous structure has not changed. Furthermore, as shown in Fig. 2c, a typical type IV isotherm with a steep capillary condensation step of IGMH nanoparticles and the surface area of IGMH nanoparticles is decreased to 211 m2/g. Specially, the pore size is about 3.4 nm (Fig. 7d). These demonstrate that Iopamidol molecules have successfully modified porous channels. CT imaging is a comprehensive and clinically commonly used imaging tools for diagnosis and medical research, in which CT contrast agents are a key factor to enhance imaging. Hence, design and research of multifunctional CT contrast agents is challenging task. In present experiments, Iopamidol was absorbed in GMH nanoparticles to form IGMH nanoparticles with bifunctional diagnosis of MRI and CT imaging. As shown in Fig. 8a, a sharp signal enhancement of CT imaging is found with the increase of IGMH concentrations, which reveals that IGMH nanoparticles are obtained and applied. Furthermore, lymphtargeted CT imaging of the dual-mode contrast agent was performed in vivo in rabbit. An attempt was made to locate lymph nodes in the knee
(Fig. 6c). Furthermore, the GMH-FITC is distributed to various organs by the blood circulation within 1.5 h (Fig. 6d). However, GMH-FITC nanoparticles focus on the kidney and brain at 2 h (Fig. 6e). With further enhancement of injection time, the content of GMH-FITC nanoparticles in the kidney and brain decreases gradually (Fig. 6f), and finally few GMH-FITC nanoparticles are remained in the kidney (Fig. 6g), which indicates that the GMH nanoparticles are likely to be cleared through the kidney. Furthermore, the result of consecutive administration monitored by ICP resulted in accumulation in liver, kidneys and spleen, meaning that these organs may be potential target organs for the of GMH nanoparticles [27], which indicate that the GMH nanoparticles could be conveyed organs through the bloodstream and finally metabolized through liver, kidneys and spleen. In order to evaluate further potential application of the material in tumor imaging, the Iopamidol is loaded on GMH nanoparticles to study the multimode imaging of IGMH nanoparticles. In Fig. 7, the morphology and structure of IGMH nanoparticles are characterized. As shown in Fig. 7a–b. The average diameter of IGMH nanoparticles is 567
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Fig. 9. T1-weighted MRI in vivo of the tumor-bearing nude mice by GMH nanoparticles at different scanning time: (a) 0, (b) 5, (c) 10, (d) 20, (e) 30 and (f) 40 min.
a quite uniform distribution of the nanoparticles within the tumor region, allowing for effective and long-periodic MR imaging of the whole tumor. In order to evaluate further potential application of the material in tumor therapy, the hydrophilic DOX is loaded on GMH nanoparticles to study the therapy function of DGMH nanoparticles. Some material characterizations are obtained and shown in Fig. 10. The surface area of DGMH nanoparticles is 213 m2/g after the mesoporous pores are filled by DOX molecules (Fig. 10b). The UV–vis absorption spectra of DOX at 0.1 mg/mL concentration of the DGMH nanoparticles and DOX are shown (Fig. 10c). The absorption spectra of DOX loading in the DGMH nanoparticles reveals characteristic absorption peaks at 490 nm, while GMH nanoparticles in UV exhibits no obvious absorption peak [28]. The drug loading of nanoparticles were 58.6% using the method of UV spectrophotometry. In order to test the drug loading properties of the materials, the release of DOX of DGMH was shown in Fig. 10d in PBS with acidic conditions (pH = 5.5) in vitro [29]. The final release amount is observed to be 30.1% at 80 h, which illustrates the substantial release of the DOX in vitro. To examine the feasibility of the obtained DGMH in biomedical applications, the targeting specificity and cytotoxicity are investigated. As is shown in Fig. 11, the cellular uptake efficiency of nanoparticles is observed by confocal laser scanning microscopy due to the red light of DOX under 488 nm exciting. It is obviously that DGMH nanoparticles are better surrounded by HCT116 cells, which is abundant expression of CD44 receptor, but few nanoparticles gathering around HEK293 cells. The cell cytotoxicity is examined to evaluate the feasibility and target ability of the DGMH nanoparticles for in vitro growth inhibitory on tumor cells. As shown in Fig. 12a and b, growth inhibition of cells is observed after incubation with free DOX and DGMH nanoparticles. The results indicate the dose-dependent cytotoxicity behavior, which reveals that the DGMH nanoparticles show remarkably higher anticancer
popliteal fossa regions both using IGMH nanoparticles (left leg) and commercial Iopamidol (right leg), which is helpful for observing the structure of the lymph nodes after IGMH nanoparticles administration. When the time is 15 min (Fig. 8b), the lymph signal intensity of injection Iopamidol (labeled with red arrow) is higher than that of IGMH nanoparticles (labeled with yellow arrow). When the time is increased from 30 to 45 min, the lymph vessels are still clear on the left and become invisible on the right (Fig. 8c–d). Furthermore, the image in Fig. 8d is reconstructed three- dimensionally to observe the content of residual contrast agents. As shown in Fig. 8e, IGMH nanoparticles in right leg (labeled with yellow arrow) is higher than that of the commercial Iopamidol in left leg (labeled with red arrow), which demonstrates that the Iopamidol inside the channels of IGMH could be released slowly. Thus, IGMH nanoparticles are a useful dual-mode contrast agent for both MR and CT imaging. In vivo CT images of IGMH nanoparticles (left leg) and commercial Iopamidol (right leg) injection in rabbit knee popliteal fossa. Scanning time after hypodermic injection: (b) 15, (c) 30, (d) 45 min, and (e) 3D Reconstruction on work-station using software. YAC-1 tumor-bearing nude mice were imaging by MR in vivo by intratumorally injection with GMH nanoparticles, which are the excellent MR imaging in vitro and targeting specificity to lymph node. That was evaluated the potential to use GMH nanoparticles for diagnosis of a xenografted tumor model. Compared to the MR image before injection (Fig. 9a), we can clearly see that the typical T1-weighted MR signal of tumor region increases obviously with injection time (Fig. 9b). With the strengthening, some subtle structures in tumor region are observed more distinctly and clearly (Fig. 9c). Moreover, the brightness of tumor region hardly decreases at 20 and 40 min post-injection, which reveals the long residence time in tumor due to the specific binding between GMH nanoparticles and their receptor (Fig. 9d–f). The results suggest that the intra-tumor injection of the GMH nanoparticles leads to 568
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Fig. 10. (a) SEM image and (b) N2 adsorption/desorption isotherm of DGMH nanoparticles, (c) UV–vis absorption spectra of DOX and DGMH nanoparticles and (d) DOX release curve from DGMH nanoparticles.
Fig. 11. Laser scanning confocal microscopy images of HCT116 and HEK293 cells incubated with DGMH nanoparticles.
efficiency on HCT116 cells than that of free DOX. On the contrary, this phenomenon could not be observed in the CCK-8 essays on HEK293 cells (Fig. 12b). We further explored the anti-tumor efficacy of the
DGMH nanoparticles in YAC-1 cell-bearing nude mice in vivo. Twelve of nude mice with YAC-1 tumor cells are randomly divided into two groups (6 rats in each group), administrated with PBS and DGMH 569
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Fig. 12. The inhibitory effect of free DOX and DGMH nanoparticles on (a) HCT116 and (b) HEK293 cells was examined by CCK-8 assay.
Fig. 13. (a) Mean tumor volume of the mice in different groups after treatment, (b) mean tumor weights of the mice in different groups after treatment, (c) photograph of the tumors from different groups after treatment. Data represent the mean standard deviation of six mice.
4. Conclusions
respectively. After 2 weeks feeding, the two groups of nude mice were intratumorally injected with 200 μL DGMH (0.1 mg/mL) nanoparticles and the same volume PBS respectively twice every week. In Fig. 13a, it is obviously that the size of the tumor for the PBS group increased significantly during the experiment. In contrast, the groups injected with the DGMH nanoparticles greatly inhibit the tumor growth, indicating the experiment. In contrast, the groups injected with the DGMH nanoparticles greatly inhibit the tumor growth, indicating the obvious anti-tumor efficacy [30,31]. After 24 days of treatment, all of the tumor-bearing nude mice were sacrificed and their tumors were taken out and weighed, the results shown in Fig. 13b indicate that the mean tumor weight of DGMH group is much lower than that of the control group. And the photograph of the tumors from different groups after treatment are also confirmed this point, (Fig. 13c). The tumor sizes of the PBS group are obviously observed bigger than that of DGMH nanoparticles group. All of the above results reveal that the DMMH nanoparticles are a powerful carrier candidate for combined diagnosis and therapy of cancer in vivo.
In summary, GMH nanoparticles are prepared by doping Gd ions to mesoporous silica material via a co-assemble process and grafting HA molecule on their surface. Furthermore, FGMH nanoparticles are obtained through the introduction of functional molecules including Iopamidol and DOX. Therefore, the active targeted nanotheranostic probe based on FGMH nanoparticles are successfully prepared and used not only as a MRI/CT contrast agent but also as an anti-tumor drug carrier. Moreover, compared with conventional contrast agent, the obtained products have potential challenge to be used as a MRI contrast due to its outstanding T1-weighted MRI signal in vivo. Specially, FGMH nanoparticles also indicate potential application in drug delivery, which presents an excellent performance in inhibiting growth of mice lymphatic tumor. Furthermore, FGMH nanoparticles are potential in high lymph targeting efficiency. Among them, IGMH nanoparticles are a dual-mode contrast agent about both CT and MR imaging for lymph system and DGMH nanoparticles are powerful agents for combined diagnosis and therapy of cancer in vivo. According to our results in the 570
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experiment, not only a new method for uniform nanoparticles but also novel and versatile FGMH nanoparticles are proposed, which could be used as an ideal candidate for simultaneous bio-imaging and chemotherapy for lymphatic system.
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