Initiation of Targeted Nanodrug Delivery in Vivo by a Multifunctional

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Initiation of Targeted Nanodrug Delivery in Vivo by a Multifunctional Magnetic Implant Jianhua Ge,†,‡ Yi Zhang,§ Zhirui Dong,†,‡ Jianbo Jia,§ Jiannan Zhu,†,‡ Xiaoyuan Miao,§ and Bing Yan*,§ †

Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials (Ministry of Education), Shandong University, Jinan 250061, China ‡ School of Material Science & Engineering and §School of Environmental Science and Engineering, Shandong University, Jinan 250100, China S Supporting Information *

ABSTRACT: Implant-mediated targeted drug delivery without an external magnetic field is very challenging. In this work, we report targeted nanodrug delivery initiated by a Fe3O4/poly(lactic-co-glycolic acid) implant scaffold with high magnetism. The implant scaffold is biocompatible and durable. It effectively attracts nanodrugs to its surface, thus killing cancer cells. These findings provide a proof of concept for the magnetic implant-directed nanodrug targeting without the need for an external magnetic field. This approach may further facilitate more precise medical treatments.

KEYWORDS: magnetic implant, drug delivery, nanocarrier, nanomedicine, targeting scaffolds can provide the field gradients for targeting only when working together with an external magnetic field.7,11 Therefore, a key challenge is to introduce a biocompatible and durable implant scaffold with sufficient magnetic strength to enable the attraction of magnetic drugs to the implant site without an external magnetic field. Magnetic nanoparticles have superparamagnetism property12 and good colloidal stability to resist aggregation in blood. These properties allow them to be driven by a magnetic field. However, a hypothesis yet to be tested is whether targeted drug delivery can be realized without the need of an external magnetic field.

1. INTRODUCTION Treatments of some devastating diseases such as broad excision in osteosarcoma or chondrosarcoma and occlusive vascular disease require medical implants. Usually, the applications of implants must be complemented by therapeutic treatment, such as providing growth factors,1 eliminating remanent cancer cells, or alleviating stent-induced injuries.2 For these treatments, magnetic scaffolds are particularly promising because controllable drug release, targeted drug delivery, or cellular fate manipulation can ideally be remotely controlled with or without an external magnetic field.3 Magnetic scaffolds have been currently prepared by in situ methods, such as dip-coating synthesis4,5 and the biologically inspired mineralization process.6 With these methods, ferric or ferrous compounds are used to produce nanoscaled iron particles on the surface of a premade scaffold, thus endowing it with magnetism. Because nanoscaled iron particles can generate only a limited magnetic force, the magnetism of the scaffolds prepared by these methods is usually low. Magnetically targeted drug delivery often requires an external magnetic field.7 However, the application of an external magnetic field during treatment is a complex and expensive proposal,8−10 especially for daily administration of therapeutics for a long duration. A possible approach to overcome this limitation is to use the magnetic field generated by an internal magnetic implant. To do so, the magnetic implants must have a sufficient magnetic field intensity to attract magnetized drugs. However, owing to the limitation of their magnetic strength, the current implant © 2017 American Chemical Society

2. RESULTS AND DISCUSSION To make a magnetic implant scaffold with a stronger magnetic strength, we first synthesized implant scaffolds H0−H3 using poly(lactic-co-glycolic acid) (PLGA) and various amounts of micron-sized Fe3O4 particles (0−40%, Table 1) by the particulate leaching technique.13−16 Scanning electron microscopy (SEM) images of the scaffold surfaces showed a uniform open-porous structure, which benefits cell adhesion and proliferation (Figure 1). The surface of H0 was rather smooth (Figure 1A, inset), whereas the surface became slightly rougher as the amount of magnetic particles increased (Figure 1B−D, Received: April 11, 2017 Accepted: May 30, 2017 Published: May 30, 2017 20771

DOI: 10.1021/acsami.7b05009 ACS Appl. Mater. Interfaces 2017, 9, 20771−20778

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ACS Applied Materials & Interfaces

diameter of the nanodrugs was 180 ± 27 nm (Figure 2B). Because of the incorporation of the fluorescence reagent, DPH, the nanodrugs were highly fluorescent (Figure 2C). The magnetic properties of the micron-sized Fe3O4 particles, the scaffolds, and 5-Fu-PLGA magnetic nanodrugs were measured with magnetic hysteresis loops by varying the magnetic field at room temperature.21−23 The micron-sized Fe3O4 particles had a saturation magnetism (Ms), magnetic remanence (Mr), and coercive force (Hc) values of 96.5 emu g−1, 18.3 emu g−1, and 137 Oe, respectively (Figure S1). Scaffold H0 (0% Fe3O4 by weight) was not magnetized at all (Figure 3A,B, the horizontal line crossing the zero point). As the Fe3O4 content increased in scaffolds H1−H3 (10, 20, and 40%), the intensity of the Ms of the scaffolds increased from 10.4 to 17, and 28.9 emu g−1 (Figure 3A,B,D). Meanwhile, the Mr (1.24, 2.0, and 3.7 emu g−1) (Figure 3A,B,D) and the Hc (136, 149, and 156 Oe) for scaffolds H1−H3 were also increased (Figure 3A,B,E). The Hc values of the magnetic scaffolds were in the range of hard magnetic materials (125−12 000 Oe), suggesting that these scaffolds might be suitable as magnetic sources.24 In contrast, 5-Fu-PLGA magnetic nanodrugs had an Hc of 2.71 Oe and Ms of 12.3 emu g−1 (Figure 3C). Such materials are classified as soft magnetic materials24 and are suitable for magnetic nanodrugs that can be guided by the magnetic field gradient. A good implant scaffold should be biodegradable to allow the implanted cells to produce their own extracellular matrix. To

Table 1. Composition of Fe3O4/PLGA Scaffolds scaffold id

Fe3O4 (w/w, %)

PLGA (w/w, %)

H0 H1 H2 H3

0 10 20 40

100 90 80 60

insets), indicating that the mingled magnetic particles increase the surface area of the scaffolds. To make nanodrugs, iron oxide nanoparticles (IONPs) were first prepared in a laboratory according to a previously reported method.17 Then, PLGA, fluorouracil (5-Fu), a well-known anticancer drug,18 and fluorescence reagent 1,6-diphenyl-1,3,5hexatriene (DPH) were mixed with the IONPs with a weight ratio of 1:1:0.5:1, and the final nanodrug was obtained by the conventional oil/water (o/w) solvent evaporation method.19 DPH was used because of its strong fluorescence when intercalated into cell membranes.20 Properties of the scaffolds and nanodrugs were then thoroughly characterized. The suspending property of 5-Fu-PLGA magnetic nanodrugs was analyzed by a laser particle size analyzer, and their morphology was analyzed by transmission electron microscopy (TEM). Laser particle size measurement showed that these nanodrugs have a hydrodynamic diameter of ∼280 nm in aqueous solution (Figure 2A), indicating that they were suspended well. TEM measurements showed that the average

Figure 1. Electronic microscopic images of the Fe3O4/PLGA scaffolds: H0 (A), H1 (B), H2 (C), and H3 (D). 20772


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Figure 2. Size measurement of fluorescent 5-Fu-PLGA magnetic nanodrugs by the laser particle size analysis (A) and TEM (B). (C) Fluorescence of the nanodrugs was observed by a fluorescence microscope. The scale bar in the inset of (B) is 50 nm.

Figure 3. Magnetization properties of the Fe3O4/PLGA magnetic scaffolds, H0−H3 (A, B, D, and E), and the fluorescent magnetic 5-Fu-loaded PLGA nanodrugs (C). The curve fragments designated by the dash box in (A) are amplified in (B) to show Hc and Mr values. Ms, Mr, and Hc of the scaffolds are plotted in (D) and (E).

maintain ideal cell viability, the stability of scaffolds must coordinate with the growth of the implanted cells, usually for 1−2 weeks.25 All scaffolds were stable in the simulated body fluid (SBF) under the physiological conditions (pH 7.4 and 37 °C) (Figure S2). To determine more quantitatively the degradation profile of the scaffolds, we measured the weight change of scaffolds after incubation with SBF. The results showed that like scaffold H0, H1 and H2 only slowly lost their weight in SBF starting from week 2 (about 1% weight loss for week 2) (Figure 4). The weight loss of H3 was about 2.1% for week 1 and 6.5% for week 2, and the weight loss of all scaffolds was less than 9% after 4 weeks. These degradation rates are comparable to those of PLGA scaffolds reported earlier.26 The degradation of scaffolds is dependent on their components.26,27 Our results indicated that the incorpo-

Figure 4. Mass changes of scaffolds incubated in SBF for various time periods.

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Figure 5. EDS analysis in a designated area of scaffold H3 after incubation in SBF for 4 weeks. (A) Analysis area is featured by a red rectangular box. (B) EDX spectrum of the featured area in scaffold H3. Inset table shows the weight and atomic ratio of different elements. (C) Histogram shows that in the deposit, the elements calcium and phosphorus show an atomic ratio of 1.6:1.

Figure 6. Cytotoxicity assay in medium that various Fe3O4/PLGA scaffolds have been immersed in for different time. The mean value from each group is shown, and the error bars designate the standard deviation from three parallel readings for each group.

ration of Fe3O4 particles in the scaffolds did not increase their biodegradation rate. These results suggested that scaffolds

incorporated with Fe3O4 particles had a desirable degradation rate and were suitable for tissue engineering applications.26 20774


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Figure 7. Attraction of nanodrugs to scaffolds and their efficacy for killing A549 cells seeded on the scaffold surface. (A) Amount of 5-Fu-PLGA nanodrugs attracted to different scaffolds after incubation for 3 days as analyzed by inductively coupled plasma atomic emission spectroscopy (ICPAES). Error bars show the standard deviations from three parallel readings for each group. (B) Cell death rate of A549 cultured on the surface of four different scaffolds after 5-Fu-PLGA treatment for 3 days.

Figure 8. In vivo image views 1, 2, and 4 days after the injection of 0.2 mL of the fluorescent 5-Fu-PLGA magnetic nanodrug suspended in phosphate-buffered saline (PBS) (1 mg mL−1) (A). (B, C) Quantification of the fluorescence intensity in the implantation sites (black circle) by Bruker MI software. The results show the mean fluorescence intensity of different regions in the circled field. Error bars are the standard variations of values of all recorded regions. (D) Percentage of the fluorescence intensity that remained around the implantation sites at different times.

designated area at week 4 (Figure 5A). The analysis revealed a higher content of C, O, and Fe, which are the components of the scaffold. The analysis also showed Ca and P were present in a ratio of about 1.6:1 (Figure 5B,C), suggesting the hydroxyapatite formation. To further test the biocompatibility and suitability of the scaffolds in the human body, we examined the scaffold-induced change in the solution pH and whether the substances that leached out of the scaffolds could cause any cytotoxicity. First, we measured the pH values of SBF at different time points and

The capability of a scaffold to bond tissues is usually evaluated by the formation of a carbonate hydroxyapatite layer on its surface in SBF, a process known as biomineralization.26−28 We next measured the biomineralization performance of the scaffolds in SBF. We found that after 1−4 weeks of incubation, significant white dots were formed on the surface of all scaffolds (Figure S3). To confirm that the white dots are hydroxyapatite (Ca3(PO4)2), H3 was analyzed by energy dispersive X-ray spectroscopy (EDS) integrated in the scanning electron microscope to detect the element composition in a 20775


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provide sufficient magnetic intensity. Because of the weak scaffolds, there is a demand for nanodrugs with a much higher magnetic strength, which is very hard to achieve. By using the particulate leaching technique, we were able to make scaffolds containing a higher amount (i.e., 40%) of Fe3O4 particles. Scaffolds of such compositions were classified as hard magnetic materials and were successfully used as a magnetic source. Under the influence of such scaffolds, the injected magnetic nanodrugs were successfully guided to the implant site. Such a unique in vivo targeting property of the scaffold was further complemented by the targeted cancer cell killing efficacy, a high biocompatibility, and the sufficient durability of the scaffold, allowing targeted disease treatment.

confirmed that the variation of pH values was less than 0.5 unit from the pH of 7.5 (Figure S4). Because the scaffolds are potentially used in tissue engineering, such as in muscle and bone regeneration, we then used osteoblasts to test the cytotoxicity. These osteoblasts come from primary bone marrow-derived stromal cells (BMSCs) after differentiation induction. The leached substance from the scaffolds did not cause any cytotoxicity to osteoblasts after a 40 h incubation (Figure 6). When osteoblasts grew on the PLGA scaffolds, the osteoblast functions were not affected.29 To test the feasibility of these scaffolds in medical applications, we next examined the magnetic field-guided drug delivery in vitro and in vivo. In suspensions containing both scaffolds and nanodrugs, we observed that more drugs were attracted to scaffolds with a higher magnetic strength (Figure 7A) after a 3-day incubation. This result indicated that the magnetic scaffold generated sufficient magnetic field to guide nanodrugs to the target site. We also examined whether the delivered drugs could kill cancer cells in vitro. The drugrelease experiment showed that 5-Fu was constantly released from the nanocarrier under physiological conditions, and 70% of the release occurred within 24 h of incubation (Figure S4). After 24 h, the concentration of 5-Fu in the solution reached a plateau (Figure S4). Then, human lung carcinoma A549 cells were seeded on the scaffolds, and the anticancer efficacy of 5Fu-PLGA magnetic nanodrugs under the guidance of magnetic scaffolds was assayed. For scaffold H0, the drug exposure led to only 20% cell death after 3 days. As the percentage of Fe3O4 particles increased in the scaffolds, cell death was gradually increased to as high as 50% for H3 in 3 days (Figure 7B). Our results indicated that our magnetic scaffolds showed excellent biocompatibility and the ability to attract nanodrugs and kill cancer cells. A key question still remained whether they could guide nanodrugs to the target site in vivo. To test this, dices (2 × 3 × 5 mm3) of H0 and H3 were subcutaneously implanted into the back of BALB/c mice (as indicated by black circles on the mouse body in Figure 8). On the following day, a fluorescent 5-Fu-PLGA magnetic nanodrug suspension (1 mg mL−1, 0.2 mL) was injected into the muscles surrounding the implantation site. The distribution of the fluorescent 5-FuPLGA magnetic nanodrugs was monitored 1, 2, and 4 days after injection. Imaging intensity quantification results showed that the injected magnetic nanodrugs in mice with the H0 implant (no magnetism) were quickly distributed to various parts of the body on days 1 and 2. On day 4, very few drugs were observed at the implantation site, and the nanodrugs were primarily found in the bladder and kidney, suggesting that the renal path was the main secretion pathway (Figure 8A,B). In contrast, in H3 implanted mice, the nanodrugs were mainly gathered around the implantation site in the first 2 days. Even on day 4, the major fraction of the magnetic nanoparticles was still accumulated around the implantation site (Figure 8A,C). These results demonstrated that the magnetic scaffold successfully guided nanodrugs to the implantation site. Bone replacement implants or implants for cardiovascular treatments have some key issues to deal with, such as recurrence of cancer growth and stent-induced injuries. It is highly desirable to automatically guide medicines to the implantation site. However, the status quo is that an external magnetic force is required to magnetize and attract the responsive drug carriers to the target site.30,31 Traditional in situ preparation methods can only produce scaffolds containing a lower amount of magnetic particles that usually cannot

3. CONCLUSIONS By developing a system of magnetic Fe3O4/PLGA scaffolds and magnetic NPs with drug payloads, we demonstrated drug delivery to the implant site and killing of cancer cells without the requirement for an external magnetic field. Furthermore, the durability and biocompatibility of the implant scaffolds indicated their potential applications as implants for the treatment of diseases such as bone cancer. The scaffolds with disease-oriented drug delivery and targeting developed in this work facilitate a more precise medical treatment. 4. EXPERIMENTAL SECTION 4.1. Chemicals. Fe3O4 powders (diameter 1−2 μm), FeCl3·6H2O, sodium oleate, alcohol, n-hexane, and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd. PLGA (lactic acid/glycolic acid in molar, 75:25; viscosity in chloroform, 0.96 dL g−1 at 25 °C) was purchased from Shandong Medical Instrument Research Institute. Oleylamine and oleic acid came from Aladdin. 5-FU and DPH were purchased from Sigma-Aldrich. 4.2. Cell Culture and Animals. All materials used for cell culture come from Invitrogen or Sigma-Aldrich. BMSCs were obtained from the bone marrow of female Wistar rats (∼180 g). BMSCs were induced for osteoblast differentiation in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 U mL−1 penicillin, 100 mg mL−1 streptomycin, 100 mM L-ascorbic acid, and 10 nM dexamethasone, as previously reported.32 A549 cells (human lung carcinoma) were purchased from ATCC and were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U mL−1 penicillin, and 100 mg mL−1 streptomycin. Female Balb/c mice (5 weeks old, ∼19 g) were housed under normal conditions with 12 h of light and dark cycles and with access ad libitum to food and water. All animal experiments were carried out in accordance with the National Institutes of Health guidelines “Guide for the Care and Use of Laboratory Animals” and experimental protocols approved by an institutional animal care and use committee. 4.3. Preparation and Evaluation of the Fe 3 O 4/PLGA Magnetic Scaffolds. 4.3.1. Preparation of the Fe3O4/PLGA Magnetic Scaffolds. Fe3O4/PLGA magnetic scaffolds were prepared by the particulate leaching technique.13−16 Briefly, PLGA was dissolved in chloroform and mixed with Fe3O4 powders. NaCl crystals (ninefolds of that of PLGA and Fe3O4 powders by weight) were added to control the porosity. The mixture was cast into a mold. After evaporation of the solvent, the scaffolds were taken out of the mold and NaCl was leached using distilled water. The ultrastructure of the scaffolds was observed by SEM (FE-SEM SU-70; Hitachi Ltd., Tokyo, Japan) after being coated with gold. 4.3.2. Magnetism Measurement of the Fe3O4/PLGA Scaffolds. The magnetic property of the Fe3O4/PLGA scaffolds was measured using hysteresis loops recorded in an alternating gradient-force magnetometer (MicroMag AGM 2900; Westerville, OH) at room temperature.21−23 4.3.3. Stability and Biomineralization Performance of the Fe3O4/ PLGA Scaffolds in SBF. SBF was prepared according to a reported 20776


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ACS Applied Materials & Interfaces method.33 The SBF used in this study has a composition similar to the human plasma (Table S1). To measure the stability in SBF, Fe3O4/ PLGA scaffolds (disk size about: 10 × 10 × 5 mm3) were incubated in 5 mL of SBF at 37 °C by shaking at 160 revolutions per minute (rpm). SBF was changed weekly for 4 continuous weeks. Before each change, the pH value of SBF was measured. The biomineralization of Fe3O4/ PLGA scaffolds was measured weekly. For weight measurement, the scaffolds were taken out from SBF, washed with distilled water, and fully dried. The surface structure of the scaffolds was observed by SEM after being coated with gold. EDS testing is performed together with SEM imaging. The FE-SEM SU-70 SEM was equipped with an HORIBA EX-250 EDS detector. The working mechanism of an EDS detector integrated in an SEM microscope has been depicted before.34 4.3.4. Cytotoxicity Assay. Scaffolds (disk size about: 10 × 10 × 5 mm3) were incubated in 1 mL of the cell culture medium in a sterile tube at 37 °C. The cell culture medium had a similar composition as that of SBF except for some nutrient components, such as proteins and glucose. Thus, the degradation rate of the scaffolds in the cell culture medium is theoretically comparable to that in SBF. After incubation for 1−7 days, the supernatant medium was taken out and used for cell culture in a 96-well plate. The osteoblasts were seeded in a 96-well plate at a density of 104 cells in 100 μL of the medium per well 24 h before treated with the supernatant from the scaffold incubation mixture. After 40 h, the cells viability was measured using the Cell Titer Assay kit using a PerkinElmer, VICTOR X2 multimode plate reader. 4.4. Preparation and Evaluation of Fluorescent 5-Fu-PLGA Magnetic Nanodrugs. 4.4.1. Preparation of Fluorescent 5-FuPLGA Magnetic Nanodrugs. For the preparation of PLGA magnetic nanodrugs, an iron-oleate complex was prepared as previously reported.35 The synthesized iron-oleate complex was used for the preparation of IONPs.17 Following this, IONPs, PLGA, 5-Fu, and DPH were dissolved in chloroform with a weight ratio of 1:1:1:0.5, and the fluorescent 5-Fu-PLGA magnetic nanodrugs were prepared by the conventional o/w solvent evaporation method.19 4.4.2. Characterization of Fluorescent 5-Fu-PLGA Magnetic Nanodrugs. The morphology of fluorescent 5-Fu-PLGA magnetic nanodrugs was observed by JEM-1011 TEM (JEOL Ltd., Tokyo, Japan). The hydrodynamic size was analyzed by a laser particle size analyzer (Zetasizer Nano ZS90; Malvern Instruments Ltd., Malvern, U.K.). The magnetic property of the fluorescent 5-Fu-PLGA magnetic nanodrugs was measured by the hysteresis loops recorded in an alternating gradient force magnetometer (model MicroMag 2900; Princeton Measurement Corp., Westerville, OH) at room temperature.21−23 For fluorescence observation, the nanodrug suspension of 1 mg mL−1 was smeared evenly on a glass slide and covered with a slipcover. The sample was mounted onto a fluorescence microscope (IX71; Olympus Corp., Tokyo, Japan) for visualization. 4.4.3. 5-Fu Release Assay. 5-Fu-PLGA magnetic nanodrug particles (16 mg) were suspended in 10 mL of PBS (pH 7.4) by stirring at 160 rpm. The drug release assay was performed at 37 °C. After incubation in PBS for 2, 4, 8, 12, 24, 36, 48, 60 h, and 14 days, 0.1 mL of the suspension was taken out. The concentrations of 5-Fu in the collected samples were determined using a UV−vis spectrometer at a wavelength of 265 nm. Because the original amount of 5-Fu in the nanodrug system is hard to determine, the released amount after incubation for 14 days was set as 100%. The drug release efficiency at different time points was calculated as a percentage relative to the total amount at day 14. 4.5. Cell Viability Analysis. The efficacy of fluorescent 5-FuPLGA magnetic nanodrugs on cell growth on the Fe3O4/PLGA scaffolds was evaluated using A549 cells. Briefly, A549 cells grew on the scaffolds in the 24-well plate (90 000 cells in 0.9 mL of the medium per well) for 24 h and then were exposed to 0.1 mL of the medium with drugs (0.5 mg mL−1). After incubation for 48 h, the cells on the scaffolds were eluted with 0.5 mL of fresh medium, from which 40 μL of the cell suspension was used for the cell viability analysis by the cell titer method. For each scaffold, three parallel wells were set up.

4.6. ICP-AES. A549 cells seeded on the scaffolds in the 24-well plate were treated with the nanodrug for 48 h. For sample collection, the scaffolds were taken out and gently washed with ddH2O three times. After that, they were vigorously pipetted with 0.5 mL of ddH2O to elute cells and the absorbed drugs. The elution process was repeated six times and all eluted suspensions were combined. To the final elute, 2.5 mL of concentrated HNO3 (65%) was added, and ddH2O was supplemented to a final volume of 10 mL. After overnight digestion, the solution was mounted for the ICP-AES (iCAP 7000 Plus Series ICP-OES; Thermo Fisher Scientific Inc., Waltham, MA) analysis. Analysis conditions: radio frequency power, 1150 W; pump speed, 50 rpm; auxiliary gas flow rate, 0.5 L min−1; nebulizer gas flow rate, 0.5 L min−1. 4.7. In Vivo Imaging. Dices (2 × 3 × 5 mm3) of H0 and H3 were subcutaneously implanted in the back of the BALB/c mice. On the second day, the fluorescent 5-Fu-PLGA magnetic nanodrug suspension in PBS (1 mg mL−1, 0.2 mL) was injected into the mice’s muscles right under the implanted scaffolds. The fluorescence images were acquired using KODAK In-Vivo Imaging System FX Pro (Carestream Health, Inc., Rochester, NY) at various time points (days 1, 2, and 4) post injection. Excitation and emission wavelengths were set as 470/535 nm, and the exposure time was set as 1 s.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05009. SEM examination of the scaffold; pH effect on the scaffold; drug release from nanocarriers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Bing Yan: 0000-0002-7970-6764 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We want to thank Shenqing Wang for help with the nanodrug preparation and Shaoshan Lian and Lin Li for animal experiments. This work was supported by the National Key Research and Development Program of China (2016YFA0203103), the National Natural Science Foundation of China (91543204 and 91643204), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030401). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

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REFERENCES

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