Abstract
Individual theranostic agents with dual-mode MRI responses and therapeutic efficacy have attracted extensive interest due to the real-time monitor and high effective treatment, which endow the providential treatment and avoid the repeated medication with side effects. However, it is difficult to achieve the integrated strategy of MRI and therapeutic drug due to complicated synthesis route, low efficiency and potential biosafety issues. In this study, novel self-assembled ultrasmall Fe3O4 nanoclusters were developed for tumor-targeted dual-mode T1/T2-weighted magnetic resonance imaging (MRI) guided synergetic chemodynamic therapy (CDT) and chemotherapy. The self-assembled ultrasmall Fe3O4 nanoclusters synthesized by facilely modifying ultrasmall Fe3O4 nanoparticles with 2,3-dimercaptosuccinic acid (DMSA) molecule possess long-term stability and mass production ability. The proposed ultrasmall Fe3O4 nanoclusters shows excellent dual-mode T1 and T2 MRI capacities as well as favorable CDT ability due to the appropriate size effect and the abundant Fe ion on the surface of ultrasmall Fe3O4 nanoclusters. After conjugation with the tumor targeting ligand Arg-Gly-Asp (RGD) and chemotherapy drug doxorubicin (Dox), the functionalized Fe3O4 nanoclusters achieve enhanced tumor accumulation and retention effects and synergetic CDT and chemotherapy function, which serve as a powerful integrated theranostic platform for cancer treatment.
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Introduction
Various well-designed functional materials have been employed as the longitudinal relaxation time (T1) or transversal relaxation time (T2) weighted MRI contrast agents (CAs) for improving image contrast and sensitivity: T1 positive agents of paramagnetic species with bright signals1,2 and T2 negative agents of superparamagnetic particles with dark signals3. Each MRI mode has its advantage, for example, T1 weighted MRI possesses excellent resolution in displaying normal soft-tissue anatomy and T2 weighted MRI possesses more outstanding in soft tissue imaging with high sensitivity to detect lesions such as tumors and inflammation. However, single MRI mode cannot meet the high diagnostic requirements owing to their inherent defects4. Since each single imaging mode has unique strengths and limitations, the utilization of advantage combinations and defect elimination of both T1 and T2 imaging technologies to increase the target-to-background signal ratio (TBR) must be urgently developed to accurately locate and diagnose lesions5,6. Paramagnetic metal ions with high electron spin angular momentum such as Mn2+, Fe3+ and Gd3+ decrease the T1 of water protons, which efficiently modify the T1 mode MRI7. The T2 is sensitive to pathological and physiological stimuli with high magnetization. Generally, researchers mainly construct T1/T2 dual-mode MRI with individual T1 and T2 contrast agents8,9,10. Unfortunately, these composite structures may suffer from elaborate synthetic procedures and unsatisfactory interference of the relaxation process. Therefore, individual contrast agents with dual-mode MRI responses have attracted extensive interest11,12.
For the design of dual-mode T1/T2 MRI contrast agent, the Fe3O4 nanoparticles with tunable MRI ability are the mostly used materials. Ultrasmall Fe3O4 nanoparticles with diameters below 5 nm have been recognized to possess T1-enhancing contrast agents due to the abundant Fe3+ on the surface of nanoparticles, while their T2 effect decreases13,14. Usually, smaller Fe3O4 nanoparticles can enhance the T1 mode detection, and larger Fe3O4 nanoparticles can improve the T2 mode image due to their large innate high magnetic moment. The small size increases the surface-to-volume ratio of Fe ions, which improves their interaction with the surrounding water protons. The decrease in the magnetic core of superparamagnetic Fe3O4 nanoparticles weakens the T2-weighted MRI response15,16. To enhance the T2 effect, self-assembled ultrasmall Fe3O4 nanoparticles were proposed to increase the magnetization. Therefore, some recent studies elaborately designed the surface chemistry of ultrasmall Fe3O4 nanoparticles to make the particle size adjustable with some bioconditions, including specific pH17,18, hypoxia19, glutathione20,21,22,23 and light24. However, some limitations still remain, such as complicated synthesis procedures, small production, and limited application conditions. Moreover, it is difficult to achieve homogeneity via the self-assembly of ultrasmall Fe3O4 nanoparticles, which affects the signal reliability. More importantly, some designs enable T1 to T2 conversion but fail to simultaneously possess both T1 and T2 effects, which urges more devotion to developing ultrasmall Fe3O4 nanoparticle-based T1/T2-weighted MRI contrast agents. Nanoparticles smaller than 5 nm can be facially cleared by the kidney, those 10–20 nm in size can be taken up rapidly, and the half-lifetime of the contrast agent in blood circulation was low25,26. The small size would make it easier for the nanoparticles to enter the tumor by permeation and retention effects, which would be beneficial for MRI examination. Therefore, the size control of the Fe3O4 contrast agent should be considered an important factor.
Integrating high bioimaging capability and therapeutic efficacy simultaneously show promising perspective for developing new effective theranostic agent27,28. The ultrasmall size of Fe3O4 nanoparticles not only endow the excellent MRI ability, but also enhance the exposed Fe ion content, which is essential for the CDT of tumor. The Fe2+ on the surface of ultrasmall Fe3O4 nanoparticles could serve as an effective catalyzer of high content H2O2 in the tumor tissue to produce poison OH·, which could effectively induce apoptosis of cancer cell. Chemotherapy, one of the most used cancer treatments in clinic, suffers from no targeting and drug resistance. The collaborative effect of ultrasmall Fe3O4 nanoparticles and chemotherapeutics could endow the MRI guided synergetic CDT and chemotherapy29,30. Moreover, integrated cancer cell targeting molecule could overcome the shortcoming of no targeting of therapeutic drugs. Therefore, the design and preparation of ultrasmall Fe3O4 nanoparticle based nanomedicine for constructing tumor-targeted imaging and treatment nanoplatform in one system hold huge promising for resolving obstacle of tumor elimination31.
In the current work, we synthesized highly crystalline ultrasmall Fe3O4 nanoparticle-based self-assembled nanoclusters with mass production for tumor-targeted dual-mode T1/T2-weighted MRI guided synergetic CDT and chemotherapy (Scheme 1). The ultrasmall Fe3O4 nanoparticles were synthesized with a size of approximately 4 nm using a solvothermal method. After modification with 2,3-dimercaptosuccinic acid (DMSA), ultrasmall Fe3O4 nanoparticles (Fe3O4-DMSA) can homogenously self-assemble into nanoclusters with several particles and maintain excellent dispersity and colloidal stability32. The Fe3O4-DMSA nanoclusters show excellent dual-mode T1/T2 MRI property due to the favorable size distribution and could effectively induce apoptosis of cancer cell by CDT. Further conjugation with Arg-Gly-Asp (RGD) and doxorubicin (DOX), RGD ligand shows an improved binding affinity toward 4T1 cells through an overexpression of αvβ3 receptors33. Dox is an effective chemotherapy drugs for cancer cell. The treatment of self-assembled ultrasmall Fe3O4 nanoclusters conjugated with RGD and DOX shows efficient T1/T2 dual-mode MRI guided tumor-targeted synergetic CDT and chemotherapy. Therefore, this work provides a novel and convenient method to prepare integrated tumor-targeted MRI and nanodrugs treatment as well as proposes the concept of nanocluster-based functional MRI contrast agents and integrated treatment platform.
Schematic of ultrasmall Fe3O4 nanoparticle-based nanoclusters for tumor-targeted dual-mode T1/T2-weighted MRI and synergetic CDT and chemotherapy.
Experiential sections
Materials
FeCl3·6H2O, sodium carbonate, sodium oleate, diphenyl ether, oleyl alcohol, doxorubicin (Dox) and 2,3-dimercaptosuccinic acid were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai). Ethanol, n-hexane, and tetrahydrofuran were purchased from Sinopharm Chemical Reagent Co., Ltd. Arg-Gly-Asp was obtained from Meilun Biotechnology Co., Ltd. (Dalian). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and sulfo-N-hydroxysuccinimide (S-NHS) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Aminofluorescein was obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Propidium iodide (PI), calcein-AM, and Cell Counting Kit-8 were obtained from Dojindo Laboratories, Japan. The Perls Stain Kit and ROS fluorescence probe (2,7-dichlorodihydrofluorescein diacetate, DCFH-DA) were from Beijing Solarbio Science & Technology Co., Ltd.
Instrument and characterization
The morphology of synthesized Fe3O4 ultra-small nanoparticles and Fe3O4-DMSA nanoclusters was characterized by transmission electron microscopy (TEM; JEM-2100, JEOL, Tokyo, Japan), and the crystal structure of the Fe3O4 nanoparticles was characterized by high-resolution transmission electron microscopy (HRTEM, JEM-2100, JEOL, Tokyo, Japan) and X-ray diffraction (XRD) patterns (D8 Advance, Bruker, Ettlingen, Germany). The element composition and atomic valence state were characterized by X-ray photoelectron spectroscopy (XPS). Fourier transform infrared (FTIR) spectra of the obtained nanomaterials were recorded by an IR spectrophotometer (Nicolet Nexus 670, Thermo Fisher Scientific, Inc., Waltham, MA). Ultraviolet–visible (UV–vis) light absorption spectra were obtained from a UV–vis spectrophotometer (UV-6100, Meipuda, Xi’an, China). The zeta potential and size distribution of the synthesized nanoparticles were characterized by a Malvern Zetasizer Nano Series. Element quantification was performed by an X-ray electron probe microanalyzer (EPMA). The ion concentration was quantified by inductively coupled plasma-mass spectrometry (ICP-MS). The saturation magnetization of Fe3O4 nanoparticles was measured by a vibrating sample magnetometer (MicroMagTM Model 2900 AGM system). The relaxation rate of MRI images of the Fe3O4 nanoprobe was tested by a 3.0-T clinical MRI scanner (GE Signa HDx 3.0 T MRI, USA) and a 16-channel brain coil.
Mass production, modification and conjugation of ultra-small Fe3O4 nanoparticles
The mass-production method of ultra-small Fe3O4 nanoparticles was adapted from a previous report34. Briefly, all iron-oleate was obtained according to previous report35. Then, iron-oleate was added to 200 mL diphenyl ether and 50 mL oleyl alcohol. The mixture was heated to 200 °C and maintained for 30 min under N2 atmosphere. The product was washed with ethanol and dried at 60 °C. The obtained brown powder was ultra-small Fe3O4 nanoparticles.
Then, the nanoparticles were modified with DMSA molecules to synthesis Fe3O4-DMSA nanoclusters. Briefly, 20 mg of Fe3O4 nanoparticles, 30 mg of DMSA, and 15 mg of Na2CO3 were added to a mixture of tetrahydrofuran and water and ultrasonicated for 30 min. Afterwards, the mixture was collected by centrifugation and freeze-dried to obtain Fe3O4-DMSA nanoclusters. Fe3O4-DMSA nanoclusters can be mass-produced by amplifying this process tween times.
To conjugate the RGD ligand to synthesis Fe3O4-RGD nanoclusters, 4 mg aqueous Fe3O4-DMSA was added to the mixture of 1 mg of RGD, 2 mM EDC, and 4 mM S-NHS and reacted at 4 °C overnight. The product was dialyzed for 24 h. To synthesis Fe3O4-RGD-DOX, additional 0.2 mg of DOX was added to the same action media as RGD and the processing procedure was the same as the conjugation of RGD ligand. The aminofluorescein conjugation procedure was identical to the RGD. The bonding mechanism between Fe3O4 and RGD as well as DOX are through the esterification between the COOH of Fe3O4-DMSA and NH2 of DOX molecule and RGD ligand.
T1 and T2 relaxivity measurement of Fe3O4-DMSA nanoclusters
The T1 and T2 relaxivity test of the Fe3O4-DMSA or Fe3O4-RGD solution were performed using a 3.0 T MR scanner (GE Signa HDx 3.0 T MRI, USA). The Fe3O4-DMSA aqueous with different concentrations (0, 2000, 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8 and 3.9 μg/mL) was measured. The following parameters were adopted in the data acquisition procedure: T1-weighted images: echo time (TE) = 20 ms; repeat time (TR) = 640 ms; FOV = 14 × 14 cm2; matrix = 256 × 256; slice thickness = 1.5 mm; spacing = 0.2 mm. (b), T2-weighted images: TE = 90 ms; TR = 4000; FOV = 14 × 14 cm2; matrix = 256 × 256; slice thickness = 1.5 mm; spacing = 0.2 mm. (c), T1 mapping: TR = 4000, 3000, 2000, 1500, 1250, 1000, 750, 500 ms; TE = 20 ms; (d), T2 mapping: TR = 3000 ms, TE = 20, 40, 60, 80, 100 and 120 ms; The reciprocal of T1 and T2 relaxation times plotted against the Fe concentrations and transverse relaxivity (r1/r2) was obtained by a linear fit.
Cell lines and animal model
4T1 (mouse breast cancer) was obtained from the Institute of Biochemistry and Cell Biology (IBCB, Shanghai, China). The cells were cultured in RPMI-1640 culture medium with 10% fetal bovine serum (Gibco, USA) and 0.1% penicillin–streptomycin (Gibco, USA). Then, they were cultured in a humidified atmosphere at 37 °C in an incubator with 5% CO2.
All animal were purchased from the Jinan Peng Yue Laboratory Animal Co., Ltd, Jinan, China. The mice were fed with a standard laboratory diet and water. All mouse research were conducted in line with protocols approved by the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine, China (accreditation number: SYXK:20190005), and all animal experiments methods were performed according to these guidelines and regulations. To develop the 4T1 bearing tumor model, BALB/c male mice (6 to 8 week-old) were orthotopically injected with 1 × 106 4T1 cells in the right hind leg.
Tumor cell targeting assay of Fe3O4-RGD nanoclusters in vitro
To verify the RGD targeting effect, Fe3O4-DMSA and Fe3O4-RGD were added to cultured 4T1 cells at 100 μg/mL for 2 h. Then, the cancer cells were fixed with 2.5% glutaraldehyde and dehydrated in 30%, 50%, 70%, 80%, 90%, and 100% ethanol. The iron element content and distribution of the cells in each group were analyzed by EPMA. Additionally, Fe3O4-DMSA and Fe3O4-RGD were conjugated with aminofluorescein and added to the cultured cells at a concentration of 100 μg/mL for 2 h. The fluorescence intensity of each group at 568 nm was observed by laser scanning confocal microscopy (LSCM, FV 300, Olympus, Japan). To verify that cellular uptake was receptor-specific, competition experiments were conducted using flow cytometry. The 4T1 cells were plated at 5 × 105 cells/ml in a 6-well plate and incubated with aminofluorescein-labeled Fe3O4-DMSA or Fe3O4-RGD solution. The cells were incubated for 12 h, washed twice with PBS, trypsinized, centrifuged and resuspended in 500 μl PBS before a flow cytometry analysis using a FACS Calibur flow cytometer (Becton Dickinson, NJ, USA).
Prussian blue staining kit were utilized to further detecte the RGD targeted effect for 4T1 cell uptake. Briefly, 4T1 cells were seeded in 24-well plates and incubated overnight. After washing with PBS twice, the cells were incubated with Fe3O4-DMSA (50 μg/mL, 100 μg/mL and 150 μg/mL) and Fe3O4-RGD (50 μg/mL, 100 μg/mL and 150 μg/mL). After 12 h cells were washed three times. Cells were fixed for 15 min in 4% paraformaldehyde, and then incubated for 25–30 min with 10% potassium ferrocyanide, rewashed twice with PBS, and counter stained with nuclear fast red for 10 min. Cells containing intracytoplasmic blue granules were defined to be Prussian blue staining positive.
Dual-mode T1/T2-weighted MR imaging ability of Fe3O4-DMSA and Fe3O4-RGD nanoclusters in vitro and vivo
For in vitro MRI, the 4T1 cells were washed with PBS and resuspended in 500 μL cell culture medium to verify the MRI effect at the cellular level by 3.0 T MRI scanner. T1-weighted imaging was performed with the following parameters: matrix size = 256 × 256, TR = 1200 ms, TE = 20 ms, slice thickness = 0.8 mm; T2-weighted imaging: matrix size = 256 × 256; TR = 2000 ms, TE = 62 ms; slice thickness = 0.8 mm. The T2-weighted signal change was calculated by using the following formula: SIi/SIc × 100% (i = 0.5, 1, 2, and 4 h), where SIc and SIi were the signal intensities of the 4T1 cells before and 0.5, 1, 2, and 4 h after incubation with Fe3O4-DMSA or Fe3O4-RGD, respectively. Additionally, the cultured cells in each group were stained with Prussian blue after 12 h of incubation with Fe3O4-DMSA and Fe3O4-RGD according to the manufacturer instructions to confirm the RGD-enhanced targeting effect.
To verify the in vivo MRI effect, 4T1 tumor-bearing mice were intravenous injection of Fe3O4-DMSA or Fe3O4-RGD (5 mg Fe/kg). T1-weighted and T2-weighted MRI of 4T1 tumor-bearing mice were acquired on a 3.0 T scanner. Scanned with the following parameters: T2 weighted imaging; FOV (field of view) = 12 × 12 cm2; matrix size = 256 × 256; slice thickness = 1 mm; TE = 60 ms; TR = 2000 ms; NEX = 2; T1 weighted imaging; FOV (field of view) = 12 × 12 cm2; matrix size = 256 × 256; slice thickness = 1 mm; TE = 10 ms; TR = 500 ms; NEX = 2 s. The MR imaging of mice were obtained at pre-injection and at 1 h, 2 h, 4 h, 8 h and 12 h post-injection. After MR scanning, slices covering the entire tumor region were prepared. TEM, ICP-MS and Prussian blue stained were utilized to verify the MRI results.
Biosafety evaluation of nanoparticles
To assess the hemolytic properties of Fe3O4-DMSA and Fe3O4-RGD nanomaterials, 2 mL of blood was dispersed to 4 mL of PBS (pH 7.4) and centrifuged at 1200 r/min for 10 min to isolate the red blood cells (RBCs). After washing with saline 3 times until the supernatant was colorless, the red blood cells were diluted with physiological saline to obtain 2% (v/v) red cell suspensions. Then, Fe3O4-DMSA and Fe3O4-RGD at different final concentrations (25, 50, and 100 μg/mL) were co-incubated with red cell suspensions for 1 h at 37 °C. Simultaneously, equal volumes of distilled water and physiological saline were selected as positive and negative controls, respectively. After the incubation, the absorbance was measured by a microplate reader at 570 nm. The hemolytic degree was expressed by the hemolytic ratio using the following formula: hemolysis ratio = (O. D of sample − O. D of negative control)/(O. D of positive control − O. D of negative control) × 100%.
Fe3O4-RGD-DOX nanomedicine for T1/T2 dual-mode MR imaging and synergistic CDT and chemotherapy in vitro
The RGD targeted CDT effect of Fe3O4 nanoclusters was furtherly studied. 4T1 cells were treated by the co-incubation of Fe3O4-DMSA (2.5, 5, 10, 20, 50, 100 and 150 μg/mL) and Fe3O4-RGD (2.5, 5, 10, 20, 50, 100 and 150 μg/mL) for 24 h. Then, the cell viability was determined using the CCK-8 assay (n = 5). The 4T1 cells were seeded in 24 cell culture plates, and a designed concentration of Fe3O4-DMSA was added to the culture medium for a specific time. Live/dead staining was performed to evaluate the cytotoxicity of the nanoclusters. Additionally, ROS staining by DCFH-DA was performed to briefly demonstrate the reason for toxicity after the 2 h co-culture of Fe3O4 at 50 μg/mL, 150 μg/mL and cancer cells.
To further verify the synergistic chemotherapy and CDT induced by Fe3O4-RGD-DOX nanomedicine, 4T1 cells were exposed to different concentrations of Fe3O4-DMSA (100 μg/mL), Fe3O4-RGD (100 μg/mL), DOX (5 μg/mL) and Fe3O4-RGD-DOX (100 μg Fe3O4/mL, 5 μg DOX/mL) and further measured by Living/dead staining and CCK-8 test. Untreated cells in the medium were used as a control. Corresponding groups without cells were used as blanks. The cell viability was calculated by assuming 100% viability in the control cells. The proliferation ability was analyzed through an EdU Proliferation Kit. Briefly, 4T1 cells grown in 96-well plates were subjected to different concentrations of Fe3O4-DMSA (100 μg/mL), Fe3O4-RGD (100 μg/mL), DOX (5 μg/mL) and Fe3O4-RGD-DOX (100 μg Fe3O4/mL, 5 μg DOX/mL) for 24 h. Then EdU was incorporated into the treated cells and detected through a catalyzed reaction with a fluorescently labeled azide and were observed by fluorescence microscopy.
To further detected the iron oxide distribution in vitro, 4T1 cells were incubated with PBS, Fe3O4-DMSA (100 μg/mL), Fe3O4-RGD (100 μg/mL), DOX (5 μg/mL) and Fe3O4-RGD-DOX (100 μg Fe3O4/mL, 5 μg DOX/mL), after 12 h. Then Prussian blue staining were utilized to show positive 4T1 cells. The stain method were performed in agreement with previously described methods. Dichlorofluorescein diacetate (DCFH-DA) was used to verify the intracellular ROS. 4T1 cells were treated with different concentrations of Fe3O4-DMSA (100 μg/mL), Fe3O4-RGD (100 μg/mL), DOX (5 μg/mL) and Fe3O4-RGD-DOX (100 μg Fe3O4/mL, 5 μg DOX/mL) for 6 h. After that, the cells were washed with PBS and incubated with DCFH-DA solution (10 μM) for another 20 min. Afterwards, the cells were washed and then observed by CLSM. Mean intracellular fluorescence intensity was analyzed with the ImageJ software.
Dual-mode T1/T2-weighted MR imaging and synergistic effect of RGD targeting CDT and chemotherapy in vivo
To confirm the effective of MRI and combination therapy of CDT and chemotherapy. T1 and T2 weighted MR imaging were detected before and after intravenous injection of 100 µL normal saline, Fe3O4-DMSA (5 mg/kg), Fe3O4-RGD (5 mg/kg), DOX (250 μg/kg) and Fe3O4-RGD-DOX (5 mg/kg Fe3O4/mL, 250 μg/kg Dox). The scanned parameters were performed in agreement with previously described methods. Coronal MRI data were collected pre-injection and post-injection.
Statistical analysis
All data were expressed as the average ± standard deviation. Statistical comparison between two groups was analyzed by the Student`s t-test. Analysis of variance (ANOVA) was used to compare the differences in different groups, and the data were defined with *p < 0.05, **p < 0.01 and ***p < 0.001.
Ethics statement
This study is reported in accordance with ARRIVE guidelines.
Results and discussions
Synthesis and characterizations of ultra-small Fe3O4 nanoparticles and Fe3O4 nanoclusters
The mass production of nearly 2 g in one reaction of the synthesized Fe3O4 nanoparticles is shown in Fig. S1, and the morphology was characterized by TEM (Figs. 1a, S2) and HRTEM (Figs. 1b, S3a). The obtained nanoparticles were of uniform shape, the size distribution was approximately 2–8 nm, and the main size was located at 4 nm (Fig. 1a, inset). The crystal plane spacings of 0.24 nm and 0.31 nm correspond to the (4 0 0) and (5 1 1) crystal planes, respectively, as shown in the HRTEM image. The well crystallinity of the individual ultra-small Fe3O4 nanoparticles is confirmed, which benefits the increased saturation magnetization. During the modification process with DMSA molecules, the Fe3O4 nanoparticles self-assemble into nanoclusters with 2–3 homogeneously embedded nanoparticles (Fig. 1c and the inset). Figure S4 confirms the stable synthesis and homogeneity of the Fe3O4-DMSA nanoclusters. The XRD pattern (Fig. 1d) of synthesized ultra-small Fe3O4 nanoparticles confirms the standard Fe3O4 phase (standard PDF card: 19-0629). The weak diffraction peak was assigned to the small size of the as-synthesized nanoparticles. Moreover, the synthesized nanoparticles were further analyzed by XPS (Fig. 1e,f). The spectrum of Fe 2p clearly shows the peaks of Fe2+ and Fe3+, which confirms the composition of Fe3O436. The spectrum of O 1s contains the peaks assigned to Fe–O, C=O, and C–O, which indicates the coexistence of Fe3O4 and oleic acid molecules. This is the key reason for the hydrophobic property of the as-synthesized Fe3O4 nanoparticles and the requirement of DMSA surface modification. The saturation magnetization curve (Fig. 1g) measured by a vibrating sample magnetometer at room temperature confirms the superparamagnetic property of the obtained ultra-small Fe3O4 nanoparticles with a large saturation magnetization (25 emu/g). The modification of Fe3O4 with DMSA molecules was studied by FTIR spectroscopy (Fig. 1h). The broad peaks at 3297 cm−1 and 3252.5 cm−1 correspond to the stretching vibrations of O–H for oleyl alcohol and DMSA, respectively. The peaks at 1516.6 and 1402 cm−1 correspond to the symmetric and asymmetric stretches of carboxylate (COO–) in oleyl acid, while the peaks at 1567.4 and 1361 cm−1 correspond to the COO– in DMSA. All C–O stretching vibrations in oleyl alcohol and DMSA are at approximately 1045 cm−1. The decreased C–H stretching vibrations at 2916.5 and 2848.1 cm−1 of oleyl alcohol indicate the substitution of DMSA at the surface of Fe3O4. Notably, the as-synthesized Fe3O4 nanoparticles are spontaneously surface-modified by the organic solvent, which contributes to the good dispersion of Fe3O4 nanoparticles in the organic solvent (Fig. S5). The surface hydrophilic treatment of nanoparticles is essential for the bio-application. XPS spectra of the Fe3O4-DMSA nanoclusters were also obtained, and the existence of S indicates surface modification by DMSA (Fig. S6). DOX molecule was confirmed to be conjugated on the surface of Fe3O4 nanoclusters in the UV–vis absorbance result to constitute the nanomedicine (Fig. 1i). The size distribution of Fe3O4-DMSA is approximately 4.5–13.5 nm and the main size locates at 6 nm. (Fig. 1j). The conjugation of the RGD ligand and DOX molecule does not change the trend of the size distribution but increases the size by approximately 2 nm (Fig. 1k). Notably, the Fe3O4-DMSA nanoclusters show excellent colloidal stability. The nanocluster suspension in water remains clear and steady after being placed at 4 °C for 10 months and the size distribution (Fig. 1l) is identical to that of before.
Characterization of ultra-small Fe3O4 nanoparticles and Fe3O4 nanoclusters. (a) TEM image of synthesized ultra-small Fe3O4 nanoparticles and the size distribution (inset). (b) HRTEM image of the ultra-small Fe3O4 nanoparticles. (c) TEM image of the synthesized Fe3O4-DMSA nanoclusters. (d) XRD pattern, (e,f) XPS pattern and (g) Saturation magnetization curve of ultra-small Fe3O4 nanoparticles. (h) FTIR spectra of Fe3O4 nanoparticles and Fe3O4-DMSA nanoclusters. (i) UV–vis patterns of Fe3O4-DMSA, DOX and Fe3O4-RGD-DOX. (j) Size distribution of Fe3O4-DMSA nanoclusters and (k) Fe3O4-RGD-DOX nanoclusters. (l) Size distribution of Fe3O4-DMSA nanoclusters after being placed at 4 °C for 10 months and a real image of the aqueous solution (inset).
Tumor cell targeting ability of Fe3O4-RGD nanoclusters
Nanomaterials usually possess passive targeting ability in tumor tissue due to the enhanced permeation and retention effect (EPR) and transport effect mediated by endothelial cells37. This ability is beneficial for the designed Fe3O4-DMSA nanocluster. However, due to the low half-lifetime of the contrast agent in blood circulation, passive targeting is limited. The active targeting ability of Fe3O4-DMSA nanoclusters is endowed by conjugation with the tumor targeting RGD ligand. It is expected to improve the accumulation of Fe3O4 nanoclusters in the tumor region and realize a favorable contrast effect. As shown in Fig. 2a, the iron element content was analyzed by EPMA after the 4T1 cells were cultured with Fe3O4-DMSA and Fe3O4-RGD for 2 h. More iron was detected in the Fe3O4-RGD group than in the Fe3O4-DMSA group, which indicates the better accumulation property of Fe3O4-RGD nanoclusters. Furthermore, the aminofluorescein molecule was conjugated with the Fe3O4-DMSA and Fe3O4-RGD nanoclusters to locate the nanoparticles by fluorescence. As shown in Fig. 2b, the 4T1 cells were cocultured with fluorescence-labeled Fe3O4-DMSA and Fe3O4-RGD nanoclusters and assayed by flow cytometry. The results convey that more cells were labeled with fluorescence in the Fe3O4-RGD group than in the Fe3O4-DMSA group, which shows that the RGD ligand enhances the cell uptake ability. Then, fluorescence-labeled Fe3O4-DMSA and Fe3O4-RGD nanoclusters were cultured with 4T1 cells at a concentration of 100 μg/mL for 2 and 4 h. As shown in Fig. 2c, after the culture with Fe3O4-RGD, the green fluorescence intensities were higher than those cultured with Fe3O4-RGD for 2 and 4 h. This result confirms the enhanced targeting effect of the RGD ligand. Prussian blue staining was utilized to further study the existence of iron in 4T1 cells after culturing with Fe3O4-DMSA and Fe3O4-RGD nanoclusters at various concentrations for 12 h. As shown Fig. 2d, the staining results are consistent with the discussion above. The tumor cells cultured with Fe3O4-RGD can accumulate more Fe3O4 nanoclusters than that cultured with Fe3O4-DMSA. These results confirm that the RGD ligand can enable an active targeting effect toward tumor cells.
Tumor cell targeting ability of Fe3O4-RGD. (a) Iron element content distribution on the surface of 4T1 cells after being cultured with Fe3O4-DMSA and Fe3O4-RGD nanoclusters (color scale bar shows the Fe element content). (b) Flow cytometry assay of 4T1 cells after incubation with fluorescence labeled Fe3O4-DMSA and Fe3O4-RGD nanoclusters. (c) Fluorescence labeling of 4T1 cells with Fe3O4-aminofluorescein and Fe3O4-RGD-aminofluorescein for 2 h. (d) Prussian blue staining of 4T1 cells after being cultured with different concentrations of Fe3O4-DMSA and Fe3O4-RGD nanoclusters for 12 h.
T1/T2 dual-mode MR imaging of Fe3O4-DMSA and Fe3O4-RGD nanoclusters both in vitro and vivo
The T1/T2 dual-mode MRI of Fe3O4-DMSA and Fe3O4-RGD nanoclusters both in vitro and in vivo were furtherly studied. As shown in Fig. 3a, for T1-weighted imaging, the phantom images show enhanced lightness with increasing Fe3O4 concentration. This result confirms that Fe3O4-DMSA nanoclusters possess the typical characteristics of T1 contrast agents. For T2-weighted imaging, the phantom images tend to be darker when the Fe3O4 concentration increases (Fig. 3b), which shows the typical characteristics of T2 contrast agents. The T1-weighted and T2-weighted MRI signals of the gradient concentrations of Fe3O4 nanoclusters indicate that the Fe3O4-DMSA nanoclusters is an excellent dual-mode T1 and T2 MRI contrast agent. The T1 and T2 relaxation times of the nanoclusters were simultaneously measured and are shown as T1 and T2 mappings according to the time horizon, respectively (Fig. 3c and d). The relaxivity values (r1 and r2) were obtained from the plots of inverse relaxation time (1/T1 and 1/T2) versus iron concentration (Fig. 3e and f). The calculated values of r1 and r2 are 0.296 mM−1 s−1 and 2.9 mM−1 s−1, respectively, and the r2/r1 value is 9.8. Generally, MRI contrast agents with a r2/r1 ratio of approximately 5–10 are determined to be T1/T2 dual-mode MRI contrast agents. Therefore, the synthesized Fe3O4-DMSA nanoclusters are excellent candidates of high-performance dual-mode contrast agents. The mechanism lies in that there still exist abundant Fe3+ ions on the surface of self-assembled nanocluster, which endow the excellent T1 mode MRI effect and the several nanoparticles self-assembled nanoclusters increase the equivalent volume of magnetic core, resulting in enhanced T2 mode MRI effect24,35.
T1/T2 dual-mode MRI of Fe3O4-DMSA and Fe3O4-RGD nanoclusters both in vitro and vivo. (a) T1-weighted MR images, (c) T1 mapping and (e) T1 relaxation time of Fe3O4-DMSA nanoclusters at various iron concentrations. (b) T2-weighted MR images, (d) T2 mapping and (f) T2 relaxation time of Fe3O4-DMSA nanoclusters at various iron concentrations. (g) T1 MRI images of 4T1 cells cultured with Fe3O4-DMSA and Fe3O4-RGD nanoclusters and (h) the corresponding signal intensity statistics. (i) T2 MRI images of 4T1 cells cultured with Fe3O4-DMSA and Fe3O4-RGD nanoclusters and (j) the corresponding signal intensity statistics. (k) T1 MRI images of tumors in vivo at different times after the injection of Fe3O4-DMSA and Fe3O4-RGD nanoclusters. (l) T2 MRI images of tumors in vivo at different times after the injection of Fe3O4-DMSA and Fe3O4-RGD nanoclusters. (m) T1- and (n) T2-weighted MRI signal intensity statistics at different times.
Based on the RGD targeting effect, the MRI effect of the designed nanoprobes were investigated on 4T1 cells before practical application in vivo. 4T1 cells were cultured with Fe3O4-DMSA or Fe3O4-RGD nanoclusters at a concentration of 100 μg/mL for 0 h, 0.5 h, 1 h, 2 h, and 4 h. Then, the cells were digested, resuspended in PBS and investigated by the 3.0 T MR scanner. The T1-weighted images (Fig. 3g) show that the T1 MRI signals became stronger with culture time for 4T1 cells cultured with either Fe3O4-DMSA or Fe3O4-RGD. Thus, more Fe3O4 accumulates in 4T1 cells during the culture. The cells cultured with Fe3O4-RGD for various times showed a higher intensity of MRI response than those cultured with Fe3O4-DMSA at the same culture time because the targeting effect of RGD on 4T1 cells enhances the accumulation of Fe3O4 nanoclusters. The statistical intensity data were analyzed as shown in Fig. 3h. The T1 signal intensities were 104%, 115.7%, 129.3% and 157% in Fe3O4-DMSA and 109%, 130%, 149% and 210.6% in Fe3O4-RGD compared with the control group after culture with 4T1 cells for 0.5 h, 1 h, 2 h and 4 h, respectively. Since the T2 mode MRI is a negative mode, the contrast agent reduces the signal. Similar to the T1-weighted images, the T2 MRI signals (Fig. 3i) become weaker with increasing culture time for both 4T1 cells cultured with Fe3O4-DMSA and Fe3O4-RGD. The cells cultured with Fe3O4-RGD for various times show a weaker intensity of MRI response than those cultured with Fe3O4-DMSA at the same culture time. These results indicate a better T2 mode MRI response with Fe3O4-RGD nanoclusters, which confirms the positive effect of the active targeting effect of RGD on 4T1 cells. The intensities are analyzed as shown in Fig. 3j. The T2 signal intensities were 93.7%, 93.5%, 86.3% and 75.6% in Fe3O4-DMSA and 78.7%, 71.3%, 53.7% and 31.6% in Fe3O4-RGD compared with the control group after culture with 4T1 cells for 0.5 h, 1 h, 2 h and 4 h, respectively. The results show that the Fe3O4 nanoclusters can actually serve as dual-mode T1/T2 MRI contrast agents to largely strengthen the contrast at the cellular level. Moreover, the RGD ligand can remarkably increase the targeting effect of Fe3O4 nanoclusters to form a faster and more high-intensity MRI response.
These Fe3O4-based nanoprobes were further investigated with 4T1 tumor-bearing mice in vivo. As shown in Fig. 3k and l, the T1 and T2 MRI images were acquired at different times after the intravenous injection of the Fe3O4-DMSA and Fe3O4-RGD nanoprobes. The effect of the contrast agent on T1- and T2-weighted images increases and decreases the MRI response, which are shown in red and blue, respectively. For the T1 signals (Fig. 3k), the signal intensity at the tumor region in both Fe3O4-DMSA and Fe3O4-RGD groups gradually increases during the first 4 h due to the rapid accumulation of Fe3O4 nanoclusters but shows a slowed increasing tendency at the later time, which may be due to the metabolism in vivo and small accumulation amount of Fe3O4 nanoclusters. At 4 h, the tumor tissue shows the best contrast to the surrounding tissues, which enables the diagnosis of the tumor tissue to be more accurate. Moreover, compared to the effect of Fe3O4-RGD with Fe3O4-DMSA, due to the targeting function of RGD to 4T1 cells, the tumor tissue of the Fe3O4-RGD group shows a higher contrast to the surrounding tissues than that of the Fe3O4-DMSA group at the same MRI examination time. Moreover, the enhanced contrast lasts longer for the Fe3O4-RGD group. Thus, the RGD targeting effect reduces metabolism in vivo. For T2-weighted imaging (Fig. 3l), the contrast enhancement of the tumor tissue was almost identical to the T1 MRI result. The main difference may be that the T2 contrast enhancement in Fe3O4-RGD is more obvious over time, which may be due to the constant aggregation effect of Fe3O4 nanoclusters at the tumor site. These MRI results in vivo are mostly consistent with the in vitro results (Fig. 3g and i). To quantitatively analyze the contrast enhancement of T1 and T2 mode MRI, the signal intensity ratios SNRpost/SNRpre at different time points were analyzed and are shown in Fig. 3m and n, respectively. The T1 signal intensities in the tumor sites were 113.8%, 118.1%, 138.1%, 121.9% and 107.3% in the Fe3O4-DMSA group and 120.8%, 136.4%, 149%, 141% and 95.6% in the Fe3O4-RGD group compared with the control group after 1 h, 2 h, 4 h, 8 h and 12 h of intravenous injection, respectively. The T2 signal intensities in the tumor sites were 94.9%, 77.7%, 75.2%, 79% and 87.3% in the Fe3O4-DMSA group and 87.4%, 72.6%, 68.5%, 66.1% and 76.5% in the Fe3O4-RGD group compared with the control group after 1 h, 2 h, 4 h, 8 h and 12 h of intravenous injection, respectively. All of results confirm that Fe3O4-RGD can serve as an efficient activatable dual-mode MRI contrast agent for precise tumor diagnosis in vivo due to the ultra-small nanoparticle-based nanocluster structure and RGD targeting effect, which reveals its tremendous potential application for accurate tumor diagnosis in the clinic.
After the injection of the Fe3O4 nanocluster-based nanoprobe, the in vivo iron distribution and biosafety were further investigated. Frozen sections of the tumor tissue were stained with Prussian blue to reveal the Fe distribution. As shown in Fig. S7, without Fe3O4 injection, no Fe nanoparticles were present in the tumor tissue in the control. Some regions stained blue due to Fe accumulation after the intravenous injection of Fe3O4-DMSA. In contrast, large areas were stained blue with the intravenous injection of Fe3O4-RGD. This result further confirms the targeting effect of RGD and the induced improved accumulation property of Fe3O4-RGD. In addition, the Fe3O4-DMSA nanoclusters are dispersed in the tumor tissue with no mass aggregation, but most of the Fe3O4-RGD nanoclusters aggregate near the blood vessels, which indicates that the RGD ligand can efficiently target the αvβ3 receptors of endothelial cells in the tumor site and tumor cells. Furthermore, ultrathin sections of tumor tissue were examined by TEM (Fig. S8). Fe3O4-based nanoclusters were detected at the tumor sites in both groups. To understand the metabolism of the Fe3O4 nanoclusters, the amount of Fe in the main organs and tumors in vivo was analyzed by ICP-MS measurements (Fig. S9). The Fe ion concentrations in the heart, spleen, lung and kidney hardly change with or without the intravenous injection of Fe3O4 nanoclusters. The Fe concentration significantly increases from ~ 100 to ~ 400 μg/g in the liver, which indicates that the liver is the main uptake organ and key metabolic organ for Fe3O4 nanoclusters. Notably, the Fe concentration in tumors increases with the application of Fe3O4 nanoclusters. Moreover, the Fe3O4-RGD group was almost 4 times the control and 3 times the Fe3O4-DMSA group. This result is attributed to the great targeting effect of RGD on 4T1 cells. Good hemocompatibility is essential for nanomaterials for biomedical applications in vivo. The hemocompatibility of the Fe3O4 nanocluster-based nanoprobe was evaluated through a hemolytic assay (Fig. S10). Compared with the effects of water (positive control) and physiological saline (negative control), no distinct hemolysis could be observed with either Fe3O4-DMSA or Fe3O4-RGD over the studied concentration range. The quantitatively analyzed hemolysis percentages of these nanoclusters were less than 2%, even when the concentration increased to 100 μg/mL These results suggest that the designed nanoprobe exhibits excellent hemocompatibility.
Fe3O4 nanoclusters based nanomedicine for T1/T2 dual-mode MR imaging and synergistic CDT and chemotherapy
Before the Fe3O4 nanoclusters being used as the MRI contrast agent and chemotherapy drug carries of tumor in vivo, 4T1 cells were firstly cultured with Fe3O4-DMSA nanoclusters at different concentrations to evaluate the CDT effect. The living/dead staining results (Fig. S11) show that there were fewer living cells with increasing Fe3O4 concentration and culture time. The CCK-8 results (Fig. S12) show the same trend of growth inhibition and toxicity effects on the tumor cells. Moreover, the cytotoxicity of Fe3O4-RGD shows distinct differences (in the concentration of 50 μg/mL, p = 0.017; 100 μg/mL, p = 0.003 and 150 μg/mL, p < 0.001) compared with Fe3O4-DMSA. This result indicates that the RGD-modified nanoparticles potentiate the CDT effect by enhancing tumor targeting effect. The ROS level in the tumor cells was detected and stained after culture with Fe3O4 nanoclusters (Fig. S13). When cultured with 50 μg/mL Fe3O4, the ROS level was much higher than that of the control. In addition, it is furtherly enhanced with 150 μg/mL of Fe3O4 nanoclusters. Therefore, the apoptosis of tumor cells induced by a high degree of oxidative stress should account for the inhibitory and toxic effects of nanoclusters. Moreover, these results indicate the abundant exposed Fe ions of the ultra-small Fe3O4 nanoparticles play a key role for the CDT of tumor cells.
After the conjugation of RGD ligand and DOX molecule on the surface of Fe3O4-DMSA nanoclusters, the integrated theranostic platform could endow the T1/T2 dual-mode MRI imaging guided synergistic CDT and chemotherapy of tumor cells. Fe3O4 nanoclusters based nanomedicine (Fe3O4-DMSA, Fe3O4-RGD, Fe3O4-RGD-DOX) were co-cultured with 4T1 cells in vitro, the EDU test was used to classify the effect of Fe3O4 nanoclusters based nanomedicine on 4T1 cells. The proliferative capacity of the treated cells (Fig. 4a) in proliferative phase were dyed green (EdU-positive) and nuclei were dyed blue. Further detailed analyses (Fig. 4c) indicate that the number of proliferating cells was least in the Fe3O4-RGD-DOX group when compared to all other groups, demonstrating that combination treatment strategy induced obvious suppressing effect on cell proliferation. Inhibitory effect in Fe3O4-RGD group on cell proliferation was more obvious than that of Fe3O4 alone (38.19 ± 3.9% vs. 61.05 ± 6.72%, *p < 0.05), indicating that RGD targeted ability was critical for combination therapy. In addition, cell proliferation in DOX group was higher than that of Fe3O4-RGD-DOX group (26.8 ± 4.4% vs. 13.2 ± 1.1%, **p < 0.01), indicating that Fe3O4-RGD-DOX suppresses cell proliferation by enhanced intracellular DOX aggregation and synergized CDT. Furtherly, the ROS staining of 4T1 cells treated with Fe3O4 nanoclusters based nanomedicines was done and shown in Fig. 4b. The statistical result of staining was shown in Fig. 4d. The results indicate that the group of DOX shows a certain degree of green fluorescence caused by oxidative stress in tumor cells. The green fluorescence rate in the group of Fe3O4-RGD was higher than group of Fe3O4-DMSA (12.99 ± 3.29% vs 20.01 ± 5.97%, **p < 0.01). This result confirms that RGD could enhance the intracellular Fe3O4 level content and the Fe3O4 nanoclusters further increase the intracellular ROS through Fenton-like reaction. Moreover, after incubation with Fe3O4-RGD-DOX, the ROS level is highest compared with other groups which indicates that Fe3O4-RGD-DOX possess synergistic effect in increasing intracellular ROS and enhancing the CDT distinctively. The CCK-8 test was also applied to assay the antitumor effect of the Fe3O4 nanoclusters based nanomedicine. As shown in Fig. 4e, the cell viability of 4T1 cells treated with Fe3O4-DMSA, Fe3O4-RGD, DOX and Fe3O4-RGD-DOX were 89.29 ± 3.32%, 67.6 ± 5.43%, 57.83 ± 2.44% and 36.47 ± 6.04% (n = 5), respectively. In addition, compared with that in the DOX group, a larger decrease of cell viability was observed in the Fe3O4-RGD-DOX group at the same concentration of DOX (P < 0.001). These results suggest that Fe3O4-RGD could synergize the DOX (chemotherapy) with RGD targeted CDT effect.
The biocompatibility of Fe3O4 nanoclusters based nanomedicine (Fe3O4-DMSA, Fe3O4-RGD, Fe3O4-RGD-DOX) in vitro. (a) EDU/DAPI staining results of 4T1 cells after coculturing with Fe3O4 nanoclusters based nanomedicine. (b) ROS staining results of 4T1 cells after coculturing with Fe3O4 nanoclusters based nanomedicine. (c) the statistical positive cell rate of 4T1 cells after being stained by EDU kit. (d) the average ROS fluorescence density statistics of 4T1 cells after being stained by DCFH-DA. (e) CCK-8 results of 4T1 cells after coculturing with Fe3O4 nanoclusters based nanomedicine.
To classify the synthesized tumor targeting effect and therapy effect, the DAPI/Actin/DOX staining (Fig. 5a) was conducted. The DAPI and the actin staining confirm the location of cell nucleus and cytoskeleton respectively. The DOX fluorescence mainly located at the cell nucleus and cytoplasm, which confirms the effective function of DOX molecule in the tumor cells. With the addition of RGD ligand, more DOX molecule could entry into cell nucleus and induce the apoptosis of tumor cells. To confirm the tumor targeted treatment of Fe3O4 nanoclusters based nanomedicine, the Prussian blue staining of 4T1 tumor cells cocultured with Fe3O4-DMSA, Fe3O4-RGD, DOX and Fe3O4-RGD-DOX. As shown in Fig. 5b, the Fe3O4-DMSA, Fe3O4-RGD and Fe3O4-RGD-DOX group show blue sediment due to the Fe distribution in tumor cells. Moreover, the Fe3O4-RGD and Fe3O4-RGD-DOX group show more obvious Fe sediment, which is due to the RGD targeting ability toward 4T1 cells. The living/dead staining were conducted and the results (Fig. 5c) indicated that the both the Fe3O4 nanoclusters and DOX could induce the death of tumor cells. Furtherly, the RGD ligand could enhance the killing effect of nanomedicine, which endow Fe3O4-RGD-DOX effective tumor targeted killing rate by synergistic CDT and chemotherapy.
Synergistic CDT and chemotherapy effect of Fe3O4 nanoclusters based nanomedicine (Fe3O4-DMSA, Fe3O4-RGD, Fe3O4-RGD-DOX) in vitro. (a) DAPI/Actin/DOX staining results of 4T1 cells after culturing with Fe3O4 nanoclusters based nanomedicine. (b) Prussian blue staining results of 4T1 cells after coculturing with Fe3O4 nanoclusters based nanomedicine. (c) Living/dead staining results of 4T1 cells after culturing with Fe3O4 nanoclusters based nanomedicine.
The MR image of tumor cell and tumor tissue endowed by Fe3O4 nanoclusters based nanomedicine was primarily conducted. As shown in Fig. 6a, the 4T1 cells were treated with Fe3O4 nanoclusters based nanomedicine and then digested, the treated tumor cells were further stained with Prussian blue. The staining result confirms the tumor targeting ability of RGD ligand and the DOX conjugation possesses no effect on the targeting effect. Furtherly, the digested tumor cells were imaged with both T1 and T2 weighted MR (Fig. 6b) and the signal statistics were shown in Fig. 6c and d. The nanomedicines induce distinctive enhancement of MRI signal intensity in both T1 and T2 MRI modal. Before injection of the designed nanomedicine, hemolysis test of Fe3O4 nanoclusters based nanomedicine was conducted. The results show that the nanomedicine possess excellent blood compatibility (Fig. S14). To confirm the MR guided combination therapy effect of CDT and chemotherapy in vivo, T1 and T2 weighted MR images were detected before and after intravenous injection of Fe3O4 nanoclusters based nanomedicine. As shown in Fig. 6e, compared with the control group, there was no significant difference in T1 and T2 imaging effects in the group of free DOX molecule before and after injection; The T1 weighted imaging signal intensity in tumor tissue increased in other groups, and the tumor tissue became brighter; For T2 imaging, the tumor tissue darkens and the signal intensity decreases. More accurately, as shown in Fig. 6f, the T1 imaging signals in Fe3O4-RGD and Fe3O4-RGD-DOX groups show significant increase (166% and 162%) compared with the Fe3O4-DMSA group (140%). As shown Fig. 6g, the T2 imaging signals in Fe3O4-RGD and Fe3O4-RGD-DOX groups show significant decrease (52% and 53%) compared with the Fe3O4-DMSA group (71%). These results indicate that RGD modified nanoclusters can enhance the effective aggregation of nanomaterials in tumor tissue through active targeted action, thereby further increasing the concentration of drugs in tumors. Meanwhile, the above results indicate that the Fe3O4-RGD-DOX nanocarrier platform can achieve visual evaluation of the treatment process through real-time in vitro MR monitoring.
Tumor targeted T1 and T2 MRI images of tumor cells and tumors as well as the signals statistic. (a) Prussian blue staining, (b) The T1 and T2 MRI image and (c), (d) the corresponding MRI signals statistic of digested 4T1 cells after incubation with Fe3O4 nanoclusters based nanomedicine. (e) The T1 and T2 MRI image and (f,g) the corresponding MRI signals statistic of tumor tissue before and after intravenous injection of Fe3O4 nanoclusters based nanomedicines.
The antitumor ability of Fe3O4-RGD-DOX based tumor targeted synergistic CDT and chemotherapy was evaluated in 4T1 tumor bearing BARB/C mice. The diagrammatic sketch of Fig. 7 detailly describes the treatment strategy. The tumor treatment results including tumor size and volume variety were shown in Fig. 7a and b. The results show that the Fe3O4-DMSA and Fe3O4-RGD groups show better tumor inhibition effect compared with the control group owing to the Fe3O4 mediated CDT in the tumor tissue. Moreover, the Fe3O4-RGD group exhibits more obvious inhibited effect compared with the Fe3O4-DMSA group (p < 0.001) owing to the tumor targeting effect of RGD ligand, which suggests that RGD mediated active targeting could enhance the CDT of Fe3O4. Compared with free DOX, the Fe3O4-RGD-DOX treated group exhibit significantly inhibition effect of tumor growth via the combinational effect from the chemotherapy and CDT. In addition, as shown in the Fig. 7c, the adopted treatment for mice would not influence the body weight, indicting the suitable dose and strategy of tumor therapy. In addition, the Prussian blue staining (Fig. 7d) and statistical result (Fig. 7h) show that the group of Fe3O4-RGD and Fe3O4-RGD-DOX have more Fe accumulation in tumor tissue. Hematoxylin/eosin (H&E) (Fig. 7e) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining with statistical result (Fig. 7f and i) show significant tumor cell death and the formation of many cavities, indicating that the Fe3O4-RGD-DOX can effectively induce tumor tissue damage. Meanwhile, the immunhistochemical studies (Fig. 7g and j) manifest that Fe3O4-RGD-DOX led to the most caspase3 expression, indicating that Fe3O4-RGD-DOX could efficiently induce tumor cell apoptosis, which well supported the in vitro anticancer result. All in all, these results indicated that the RGD meditated tumor targeted synergistic treatment effect of chemotherapy and CDT induced by ultrasmall Fe3O4 nanoclusters and DOX hold great tumor-growth inhibition effect.
In vivo antitumor and biosafety evaluation of tumor targeted synergistic CDT and chemotherapy. (a) Real tumor image after the collaborative treatment. (b) Tumor volume of tumor bearing mice during the collaborative treatment. (c) Body weight of tumor bearing mice during the collaborative treatment. (d) Prussian blue staining, (e) HE staining, (f) TUNEL staining, (g) Caspase 3 staining of tumor tissue sections after the collaborative treatment. (h) The statistics of Fe ion expression obtained from Prussian blue staining (i) the statistics of positive expression of TUNEL. (j) The statistics of positive expression of Caspase 3.
Biosafety evaluation after synergetic theranostics
After the in vivo antitumor study, the H&E staining of main organs including heart, liver, spleen, lung and kidney were used to evaluated biocompatibility and histological variations of Fe3O4 nanoclusters based therapy. As shown in Fig. 8a, the sections from all organ tissues in Fe3O4-DMSA, Fe3O4-RGD, DOX and Fe3O4-RGD-DOX groups demonstrated no histological difference compared with the control group. In addition, no histopathological damage was observed, including necrosis, swelling, or inflammatory response. Good hemocompatibility is also essential for the application of nanomaterials in biomedical applications in vivo. The hemocompatibility of the Fe3O4 nanocluster-based nanomedicine was evaluated through blood test. Moreover, the main blood indexes of mice treated with Fe3O4-RGD-DOX as well as other treatment at different days were further analyzed, showing no obvious change in all groups (Fig. 8b–e). These preliminary results show that the designed nanomedicines possess excellent biocompatibility in vivo.
Biosafety evaluation of collaborative treatment of Fe3O4 nanoclusters based nanomedicine. (a) Images of hematoxylin and eosin (H&E)-stained histological tissue sections of major organs. (b–e) Main blood indexes of mice during the collaborative treatment.
Conclusion
In summary, an effective method of mass produce ultra-small Fe3O4 nanoparticles was proposed. The further surface modification with DMSA endows the hydrophilic property and high colloidal stability in suspension as well as the self-assembled property. The obtained Fe3O4 nanoclusters show an excellent T1/T2 dual-mode MRI capacity and excellent potential for use as contrast agents due to the appropriate size effect. In addition, the ultrasmall Fe3O4 nanoclusters possess notable CDT effect caused by the increased ROS level in tumor cells. The collaborative work of chemotherapy drugs DOX and Fe3O4 nanoclusters base CDT treatment endow highly effective antitumor activity for tumor treatment. With the assistance of RGD targeting ligand, the integrated Fe3O4-RGD-DOX nanoplatform possesses tumor targeted T1/T2 dual-mode MRI guided synergistic CDT and chemotherapy, which provides a novel practical alternative for integrated diagnosis and treatment of tumors and exhibits promising potential for clinical translation.
Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
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Acknowledgements
This work was supported by the Key Research and Development Program of Shandong Province (2021CXGC010603), Shandong Provincial Natural Science Foundation (ZR2023QE146), the Interdisciplinary Science Innovation Group Project of Shandong University (2020QNQT001).
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Q.H.X. and X.M.W. performed the experiments, collected the experiment data and completed the manuscript. G.R.Z., D.W.Z. and Y.X.Z. helped handling the MRI machine and MRI data. J.Z.D gave a detailed guidance and experiment support for this project. H.L., D.X.Y. and Y.H.S. designed this program and proposed an elaborate guidance for this project.
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Xie, Q., Wang, X., Zhang, G. et al. Ultrasmall Fe3O4 nanoparticles self-assembly induced dual-mode T1/T2-weighted magnetic resonance imaging and enhanced tumor synergetic theranostics. Sci Rep 14, 10646 (2024). https://doi.org/10.1038/s41598-024-59525-2
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DOI: https://doi.org/10.1038/s41598-024-59525-2
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