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An injectable and self-healing thermo-sensitive magnetic hydrogel for asynchronous control release of doxorubicin and docetaxel to treat triple-negative breast cancer Wensheng Xie, Qin Gao, Zhenhu Guo, Dan Wang, Fei Gao, Xiu-Mei Wang, Yen Wei, and Lingyun Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10699 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017
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An injectable and self-healing thermo-sensitive magnetic hydrogel for asynchronous control release of doxorubicin and docetaxel to treat triplenegative breast cancer Wensheng Xie†,‡ Qin Gao†,‡, Zhenhu Guo†,‡, Dan Wang†,‡, Fei Gao†,‡, Xiumei Wang†,‡, Yen Wei#*, Lingyun Zhao†,‡*
†
State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science
and Engineering, Tsinghua University, Beijing 100084, China
‡
Key Laboratory of Advanced Materials, Ministry of Education of China, School of
Materials Science & Engineering, Tsinghua University, Beijing 100084, China. E-mail:
[email protected]
#
Department of Chemistry, Tsinghua University, Beijing 100084, China. E-mail:
[email protected]
KEYWORDS
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Co-delivery system; Chemotherapy; Magnetic hydrogel; Multidrug resistance; Control release; Breast cancer;
ABSTRACT
Integration of two or more drugs into a multi-agent delivery system has been considered to have profound impacts on both in vitro and in vivo cancer treatment due to their efficient synergistic effect. This study presents a cheap and simple chitosan hydrogel crosslinked with telechelic difunctional poly (ethylene glycol) (DF-PEG-DF) for synthesis of an injectable and self-healing thermo-sensitive dual-drug-loaded magnetic hydrogel (DDMH), which contains both doxorubicin (DOX) and docetaxel (DTX) for chemotherapy, and iron oxide for magnetic hyperthermia induced stimuli responsive drug release. The as-prepared DDMH not only have good biocompatibility but also exhibit unique self-healing, injectable, asynchronous control release properties. Meanwhile, it shows an excellent magnetic field responsive heat-inducing property which means that DDMH will produce a large amount of heat to control the surrounding temperature under the alternative magnetic field (AMF). A remarkably improved synergistic effect to triple negative breast cancer cell line is obtained by comparing the therapeutic effect of co-delivery of DOX and DTX/PLGA nanoparticles (DTX/PLGA NPs) with DOX or DTX/PLGA NPs alone. In vivo results showed that DDMH exhibited significant higher antitumor efficacy of reducing tumor size compared to single drug-loaded hydrogel. Meanwhile, the AMF-trigger control release of drugs in co-delivery system has a more efficient antitumor effect of cancer chemotherapy, indicating that DDMH was a promising multi-agent co-delivery system for synergistic chemotherapy in the cancer treatment field.
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1. INTRODUCTION
As one of the most severe or even life-threatening diseases, cancer has become a major public health issue worldwide. 1 , 2 Despite the tremendous and continuous on-going advances in improved or novel treatment to fight the deadly disease,3,4 however, the solid tumor still remain to be difficult to cure effectively.5,6 Currently in cancer clinical, surgical treatment of the primary tumors and/or adjacent lymph nodes is by far the first choice and most adopted approach for the resectable carcinoma.7 In attempt to prevent the treatment failure caused by local recurrence or metastasis of primary tumor, post-surgical treatment is immediately followed up.8 As an adjuvant therapy for cancer surgical resection, chemotherapy plays a vital role in effective cancer management.9,10 Except for development new chemotherapeutic agents with novel mechanisms of action, higher cytotoxicity or reduced toxicity, the antitumor effect can often be enhanced by improvement in the ways of drug administration. Based on the fact that the anticancer agents normally exhibit a rather steep dose-response for toxicity and therapeutic effects, localized chemotherapy, as compared with systematic drug administration, can be a more effective delivery strategy.11,12 Such local therapy system is usually designed to implant immediately at the tumor resected cavity after tumor debunking surgery, to detriment the malignant cells which may have survived surgery. By direct administering the drug-loaded implants at the tumor site, the localized thermotherapy can offer the advantages as prolonged and controlled drug release directly to the tumor tissues, so as to ensure adequate diffusion and uptake by the cancer cells over many cycles of tumor cell division.13 More importantly, such protocol can effectively diminish the side effect due to the avoidance of systemic circulation of the chemotherapeutic agents.14 Therefore, local delivery system for direct drug release is thus highly desired for post-surgical treatment.
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As advanced by polymer-based drug delivery depots, numerous formulations on drug-loaded implants have been designed and synthesized, normally for single drug-induced cancer localized chemotherapy in the past few decades, such as polymer millirods,15 flexible film composites, 16 hydrogels 17 , 18 and expansile nanoparticles. 19 However, due to the intrinsic heterogeneity of the tumor tissues and the differences in cancer cell divisions or growth stages, single drug strategy may fail to kill all the cancer cells.20,21 Therefore, the synergistic combination of two or more chemotherapeutic drugs with different toxicity profiles and cytotoxic action mechanisms is highly desired for cancer chemotherapy. 22
, 23
More
importantly, such combination chemotherapy may also be able to delay the generation of the so called multi-drug resistance (MDR), so as to remarkably improve the therapeutic effect.2426
In recent years, combination chemotherapy has revealed a high anti-tumor efficacy. Hai
Wang reported a kind of core-shell NPs which were doubly emulsified using an amphiphilic copolymer methoxy poly (ethylene glycol)-poly (lactide-coglycolide) for co-delivering hydrophobic paclitaxel and hydrophilic doxorubicin.27 The results showed that compared with the delivery of paclitaxel or doxorubicin alone, the co-delivery system could suppressed the growth of tumor cells more efficiently with the same concentrations. Shixian Lv has successfully designed a nanovehicle based on an amphiphilic deoxycholate decorating methoxy poly (ethylene glycol)-b-poly (L-glutamic acid)-b-poly (L-lysine) triblock copolymer for the co-delivery of chemotherapeutic agents.28 In vitro cytotoxicity assay using human lung adenocarcinoma cell (A549) showed that the paclitaxel and doxorubicin codelivered nanoparticles exhibited a synergistic effect by inducing apoptosis of cancer cells. These co-delivery systems which deliver different therapeutic drugs with various physiochemical
and biological properties to the in vitro and in vivo tumor cells
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simultaneously, have been proved to be successful with respect to minimize the administration dosage of drug and achieve the synergistic effect after a single injection. However, the releasing behaviour of different chemotherapeutic agents in these co-delivery systems (like doxorubicin and docetaxel, doxorubicin and paclitaxel) was synchronous. This strategy will fail when the recurrence of cancer cells happens or the residual tumor appears after a single injection.
29
In addition, a controllable and sustainable release of
chemotherapeutic agents stimulated by mediators, for example iron oxide nanoparticles which could produce much heat under an alternative magnetic field to speed up the diffusion rate of agents, could further improve the therapeutic effects. 30 Therefore, a co-delivery system which could release therapeutic agents asynchronously stimulated by physical or chemical methods, has a significant potential to solve the influence of MDR and residual tumor of cancer therapy at a low dosage of drugs for the synergistic effect.
Currently, docetaxel (DTX) and doxorubicin (DOX) have been considered the most crucial chemotherapeutic agents used in oncology clinic for cancer management.31-33 While DTX can act as an effective microtubule-targeting agent for the covalent binding to assembled tubulin, so as to promote and stabilize persistent interphase microtubule complexes, 34 DOX can function as a DNA-intercalating agent to further inhibit RNA and DNA biosynthesis.35,36 Although some studies have demonstrated that the co-delivery of DOX and DTX could increase the tumor regression rate compared to the individual drugs, but the co-delivery of DOX and DTX which could achieve both synchronous delivery and asynchronous control release was rarely investigated. Therefore, it is of significant importance to design an effective and simple co-delivery system to obtain the goal.
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In the present study, in order to co-deliver multi-antitumor drugs within a simple nanocarrier with synchronous delivery and asynchronous control release performance, we proposed a cheap and simple chitosan hydrogel crosslinked with telechelic difunctional poly (ethylene glycol) (DF-PEG-DF) for synthesis of dual-drug-loaded magnetic hydrogel (DDMH) which contain both doxorubicin and docetaxel for synergistic chemotherapy, and iron oxide for magnetic hyperthermia induced stimuli responsive drug release. Due to its unique injectable, self-healing, asynchronous control release properties, DDMH is a promising multiagent codelivery system for synergistic chemotherapy of breast cancer.
2. MATERIALS AND METHODS
2.1. Materials. Glycol chitosan (CS, 430 kDa) with 75.2% deacetylation was purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). DF-PEG-DF was synthesized by Yaribo Co. Ltd. Ethanol, ethylene glycol (EG), diethylene glycol (DEG), sodium acetate anhydrous (NaAc), sodium dodecyl benzene sulfonate (SDBS), and dichloromethane (DCM) were supplied by Sinopharm Group Co. Ltd. Polyvinyl alcohol (PVA) and iron(III) chloride hexahydrate (FeCl3·6H2O) were obtained from Sigma-Aldrich Co. LLC. (St. Louis, Missouri, USA). Poly (lactic-co-glycolic acid) (PLGA) was obtained from Jinan Daigang Biomaterial Co. Ltd. Doxorubicin (DOX) and docetaxel (DTX) were purchased from Shanghai Jinhe Bio-Technology Co. Ltd (Shanghai, China). High-glucose dulbecco’s modified eagle’s medium (H-DMEM), RPMI 1640 medium (RPMI 1640), penicillinstreptomycin (PS), and fetal bovine serum (FBS) and trypsin were provided by Gibco Life Technologies (Beijing, China). Cell Counting Kit-8 (CCK-8 assay) and were supplied by Dojindo Molecular Technologies Inc. (Kumamoto, Japan). Deiomized (DI) water purchased
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from Sinopharm Chemical Reagent Beijing Co. Ltd. was used throughout. All materials were used without further purification.
2.2. Preparation of Docetaxel-loaded PLGA nanoparticles (DTX/PLGA NPs). DTX/PLGA NPs were synthesized via a modified solvent extraction/evaporation method according to our previous publication. 37 Briefly, 110mg PLGA and 10mg DTX were dissolved in 8 ml of dichloromethane solution (DCM). The formed organic solution was pipetted dropwise into 120 mL 5% (w/v) polyvinyl alcohol (PVA, as surfactant) DI water solution with gentle stirring. Then the mixture solution was sonicated with a sonication power of 300W for 3 min. To remove the DCM, the formed mixture solution was evaporated overnight with vigorously magnetic stirring. The particle suspension was collected by centrifuging at 12,000 rpm for 10 min and washed three times with DI water to remove the unloaded DTX and the surfactant. Finally, the resulted products were resuspended in 10 ml DI water and then freeze-dried for 48 h.
2.3. Synthesis of DF-PEG-DF modified iron oxide magnetic nanoparticles (Fe3O4@DFPEG-DF MNPs). Fe3O4 MNPs were synthesized via a hydrothermal method, in which Fe3+ was partially reduced before the formation of Fe3O4 MNPs. Generally, the reactants of 1.35g FeCl3·H2O, 2.325g NaAc and 1.4g SDBS were consequently added into the mixture of 10 mL EG and 30 mL DEG. The as-prepared mixture was then under continuous stirring for 5 h and sealed in a 50 ml Teflon-lined stainless-steel autoclave (with a filling ratio about 70%) at 200℃ for 12h. After natural cooling down to room temperature, the particles were washed with ethanol and DI water respectively for three times, then collected by magnetic separation and dried in a vacuum oven at 60℃ for 24h. One-pot synthesis protocol was applied for the
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purpose of modification of DF-PEG-DF onto the MNPs surface by adding 0.2g DF-PEG-DF into the mixture of EG and DEG during the particles synthesis as above-mentioned.
2.4. Synthesis of dual-drug-loaded magnetic hydrogel (DDMH). Doxorubicin (DOX), DTX/PLGA NPs and together with Fe3O4@DF-PEG-DF were dispersed within the 3% (w/v) chitosan-NH2 aqueous solution. The as-prepared suspension was then mixed with 2.75% (w/v) DF-PEG-DF solution with the ratio of 4:1 (v/v). The mixture was stirred immediately for a few seconds and then stewed to form the DDMH.
2.5. Characterizations of the DTX/PLGA NPs, Fe3O4@DF-PEG-DF MNPs and DDMH. 1
H NMR spectrum was recorded on a JEOL (JNM-ECA300, JEOL, Japan) 300 MHz
spectrometer. The morphology and size of the as-prepared samples were observed on a fieldemission scanning electron microscope (FE-SEM, S-4800, HITACHI, Japan) and transmission electron microscope (TEM, HT7700, HITACHI, Japan). Hysteresis loop was measured with a SQUID MPMS XL-7 magnetometer. The temperature change profiles of Fe3O4@DF-PEG-DF MNPs and DDMH under an alternative magnetic field (AMF) was measured by an inductive heating device (Shuangping Instrument Technology, Co., Ltd., Shenzhen, China) and temperature was recorded by optical fiber probe (ThermAgile-RD, Xi'an Heqi Opto-Electronic Technology Co., Ltd., Shanxi, China).
2.6. Rheological studies. A Rheometer AR1000 (TA instruments, New Castle, DE, USA) was applied to study the rheological properties and self-healing performances of the asprepared hydrogel with a 20-mm parallel-plate configuration. A strain step cycled between 0.001% and 10% at 25℃ and 10 rad/s angular frequency, and a frequency step cycled between 1-100 rad/s at 25℃ and 1% strain were performed. The changes of storage (G’) and
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loss (G’’) modulus with strain and angular frequency were recorded, respectively. A temperature cyclic step test varying between 0℃ and 37℃ with angular frequency of 10 rad/s and 1% strain was performed. The changes of G’ and G’’ with time was recorded. To characterize the self-healing performance of the DDMH, external strain increasing from 0.1% to 1000% was applied to a 10 wt% sample at 37℃ and rad/s, followed by an immediate return of strain to 1%.
2.7. Injectable and self-healing behaviours of DDMH. To demonstrate the injectable property of DDMH, a 1 mL/cc syringe was used to extrude the DDMH through the 26-guage needle (Φ = 260µm). For self-healing behaviour, DDMH were prepared in a round 48-well plate and the aged in the mould at ambient temperature for 1 h. Then the as-prepared DDMH was injected above the glass beads in an EP tube and placed in room temperature. Photos were taken at 0, 1 and 2 h.
2.8. In vitro 3D culture and biocompatibility. L-929 cells (~1.0 x 106 cells/per mL) were embedded by DDMH and after gelation (1.0 mL), the cell-laden constructs were for another 48 h incubation. The cell culture medium was replaced every day. Before the analysis of cell proliferation, the cell-laden constructs were washed with phosphate buffered saline (PBS) twice and stained with Calcein-AM and PI for 20 min. Laser Scanning Confocal Microscope (LSCM, Zeiss, LSM780) was used to determine the distribution of living and dead cells in hydrogel.
2.9. In vitro drug release assay. 1 mL of various kinds of hydrogels with different components, including DOX loaded hydrogel (DOX hydrogel), DTX/PLGA NPs loaded hydrogel (DTX/PLGA NPs hydrogel), dual drug loaded hydrogel (DOX+DTX/PLGA NPs
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hydrogel), and DDMH was put into pouch made of regenerated cellulose dialysis membrane, which was soaked into release buffer (PBS containing 0.1% w/v Tween 80, pH 7.4) and incubated in 37oC water bath with 200 rpm shaking. Tween 80 was used to improve the solubility of DTX in the PBS buffer solution and eliminate the binding to the tube wall. At given time intervals, the release buffer was sampled for DOX and DTX measurement and then replaced with fresh buffer. The amount of DOX was calculated by recording the UV-visNIR spectra using a microplate reader (Varioskan LUX, Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) according to the standard curve of DOX. For DTX analysis, 1 mL release buffer was extracted by 2 mL DCM. Then, the DCM was evaporated and the solutes was dissolved with MeOH and subjected to HPLC analysis. For the HPLC assay, the mobile phase consisted of DI water and acetonitrile (50:50, v/v) was applied. A reverse-phase Inertsil_C-18 column (4.6 x 250 mm, pore size 5 mm, GL science Inc., Tokyo, Japan) was used for separation. With a 1 mL/min flow rate of mobile phase, the effluent was detected by a UV/VIS detector at 227 nm. In the range of 50–50,000 ng/ml, the calibration curve was linear with a correlation coefficient of R2 =0.9999.
For AMF-triggered release of drugs from the DDMH, 1 ml of DDMH which bag was subject the exposure under the AMF (19.33kA/m) for 10 min at designated time intervals and then put back into the shaker for incubation.
In vitro cytotoxicity assays. CCK-8 assay was performed to test cell toxicity of hydrogels with different components on MDA-MB-231 cells. Briefly, cells in the exponential phase were seeded in 24-well plate at a density of 2.5 x 104 cells/well. After 24 h incubation, 0.5 mL hydrogel were added into each 4-transwell insert and the insert was then placed into the cell containing well. Additionally, 0.5 mL of culture medium () was added. The cell was
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subjected to CCK-8 assay after 48 h co-incubation with hydrogels with different components. For CCK-8 assay, the absorbance value was determined by the spectrophotometry of microplate reader at 450 nm. The results were showed as the relative percentage of cell viability compared with the untreated cells.
In order to investigate the drug synergistic effect in hydrogel-based co-delivery system, a combination index (CI) analysis was conducted. The dose-effect profile against the concentrations of drug in a logarithmic scale was created and the sigmoidal curve-fitting analysis was made for calculation. The inhibitory concentration values (ICx) whose definition is the concentration of a certain drug which produce x% cell inhibition to certain cell line, were determined through the sigmoidal equation. CI values which is provided by the doseeffect profile under a given drugs combination, were plotted in terms of the ICx values. CI analysis was used to determine the extent of drug interactions according to Chou and Talalay’s method.38
CIx = D1/D1x + D2/D2x
Where D1x and D2x represent the ICx value of drug 1 and 2 alone, respectively. D1 and D2 is the median effect dose in the combination system of drug 1 and 2 at the ICx value. With CI < 1 demonstrating synergism, CI = 1 demonstrating additive and CI > 1 demonstrating antagonism, the equation directly provide the quantitative information of the extent about the two drug interactions. ified
2.11. Toxicology evaluation of DDMH. Healthy Balb/c female mice were subcutaneously injected with 200 µL DDMH and sacrificed at 1, 3, and 12 weeks after injection for
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toxicology evaluation. An approximate 1.0 mL portion of blood obtained from each mouse was drawn before scarification for a complete blood panel analysis and blood chemistry test. The serum chemistry data and complete blood panel were measured in the Hospital of Tsinghua University. Major organs including skin, heart, liver, spleen, lung and kidney were then taken and fixed in 4% neutral buffered formalin. After being processed routinely into paraffin and sectioned at 8 µm, a digital microscope (Leica QWin) was used to examine the hematoxylin and eosin (H&E) stained slices. Statistical analysis were based on the standard deviation of 3 mice in each group.
2.12. In vivo anti-cancer efficacy. All the animal experiments were conducted according to the guidelines for laboratory animals established by the Laboratory Animal Research Center, Tsinghua University. Female balb/c mice were subcutaneously injected with MDA-MB-231 cells at a concentration of 107 cells/mouse. After the tumor volume reached to 100 mm3, mice were divided into 5 groups randomly (n = 5): Saline group (intratumoral (i.t.) injected with 100 µL saline solution), DOX Hydrogel group (i.t. injected with 100 µL DOX loaded hydrogel (with a DOX concentration of 0.5 mg/kg)), DTX/PLGA NPs Hydrogel group (i.t. injected with 100 µL DTX/PLGA NPs loaded hydrogel (with a DTX/PLGA NPs concentration of 68.5 mg/kg)), DOX + DTX/PLGA NPs Hydrogel group (i.t. injected with 100 µL both DOX and DTX/PLGA NPs loaded hydrogel (the amount DOX and DTX/PLGA NPs is equal to DOX group and DTX/PLGA NPs group), DDMH group (i.t. injected with 100 µL DDMH (the amount DOX and DTX/PLGA NPs is equal to DOX +DTX/PLGA NPs group and the concentration of Fe3O4@DF-PEG-DF MNPs is 18.7 mg/kg). 24 h later, the DDMH group were treated under a AMF for 10 min with an intensity of 19.99 kA/m (the frequency is 282 kHz). Meanwhile, the temperature changes of tumor site during laser irradiation was monitored using an IR thermal camera. The tumor sizes and body weights
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were measured and recorded every 2 days with a digital caliper and electron balance. The tumor volumes were calculated by: volume = width2 × length/2. Relative tumor volumes were calculated as V/V0 and relative body weight were calculated as W/W0 (V0 and W0 were the tumor volume and body weight when the treatment was initiated, respectively).
2.13. Statistical analysis. All data were presented as the mean ± standard error (S.E.). Statistical analysis was conducted using one-way ANOVA followed by a Newman–Keuls post hoc t-test with the SPSS (IBM7® SPSS® Version 23) for windows. P-values which were less than 0.05 were considered statistically significant.
3. RESULTS AND DISCUSSION
3.1. Preparation and Characterization of DTX/PLGA NPs, Fe3O4@DF-PEG-DF MNPs and DDMH. DTX/PLGA NPs were successfully prepared by the single emulsion method. The SEM images were shown in Figure S1A1 (Supporting Information), which reveals that the DTX/PLGA NPs are spherical or almost quasi-spherical in shape with narrow size distribution of 210 nm, which was further confirmed by the TEM image (Figure S1A2 (Supporting Information)).
Drug loading ratio and encapsulation efficiency of DTX in
DTX/PLGA NPs is 14.8% and 95.6 %, respectively. Before the modification of Fe3O4 MNPs with DF-PEG-DF, DF-PEG-DF was measured by
1
H NMR. The result (Figure S2
(Supporting Information)) showed that the ether methylene protons of polymer backbone showed signals at 3.63 ppm, and new peaks were corresponded to the ester methylene groups of 4.50 ppm, the benzene ring groups of 8.22 and 7.97 ppm and the aldehyde groups of 10.02 ppm, respectively. The integration ratios among 8.22, 7.97, 4.50 and 3.63 were 2/2/2/182, which is significantly close to the theoretical value of 2/2/2/174, demonstrating that all the
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polymer chains at both ends were terminated with benzaldehyde groups. The morphology of as-synthesized Fe3O4@DF-PEG-DF MNPs was shown in Figure S2 (Supporting Information), most of the nanoparticles are spherical with sizes about 75 nm. The TEM images revealed that the surface of Fe3O4@DF-PEG-DF MNPs ((Figure S3A (Supporting Information))) were similar compared with Fe3O4 MNPs ((Figure S3B (Supporting Information))), which indicated the successful modification of DF-PEG-DF and the surface modification of DF-PEG-DF onto the Fe3O4 MNPs would not change the morphology of the MNPs.
Both DTX/PLGA NPs and Fe3O4@DF-PEG-DF MNPs were freeze dried and the assynthesized powders were redispersed within 3% (w/v) chitosan-NH2 aqueous solution and then mixed with 2.75% (w/v) DF-PEG-DF solution with the ratio of 4:1 (v/v). A multicomponent hydrogel thus can be facile prepared within a short gelation time of ~150 s. As demonstrated in Scheme 1, the hydrogel network is created by the dynamic covalent Schiff-base linkage between the benzaldehyde groups at both DF-PEG-DF chain ends and the NH2 groups within the chitosan-NH2 molecules. Because of the intrinsic property of Schiffbase linkage’s dynamic equilibrium, which could act as a quasi-covalent linkage, the continuous regeneration and cleavage of the imine bond within the hydrogel network thus indicates that the hydrogel can demonstrate self-healing and self-adaptive property automatically without additional stimuli.
Currently, with the active response behaviors and extensive applications, magnetic hydrogel has emerged as a novel bio-composite and different protocols were developed for the fabrication of magnetic hydrogel. Many focuses have been paid on the blending method and in situ precipitation method. while the former one aims at dispersing the prepared MNPs sediment with aqueous or oil phase and then mixed with hydrogel precursor solution to
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encapsulate the MNPs within hydrogel, and the later one takes the hydrogel matrix as a chemical reactor, where the inorganic iron ions which swollen in hydrogel networks could react with precipitating agents (e.g. NaOH, NH3 · H2O) to generate MNPs within hydrogel matrix. For both above-mentioned methods, no covalent bonds are formed between the hydrogel networks and the MNPs and therefore, the homogenous distribution and stability of MNPs within the hydrogels cannot be guaranteed. In the current study, the surface coating of DF-PEG-DF onto the Fe3O4 MNPs surface can enable the Fe3O4@DF-PEG-DF MNPs could act as the nano-crosslinkers to form the covalent coupling with glycol-chitosan. Such grafting-on strategy can realize the uniform MNPs distribution within the hydrogel matrix and also guarantee the stability of the as-prepared magnetic hydrogel for the precipitation and aggregation of MNPs was blocked, 39 as illustrated by Figure S4 (Supporting Information).
The injectable and self-healing DDMH which has intrinsic self-healing properties produced by the dynamic covalent chemistry between amimo group and phenyl aldehyde group was evaluated as shown in Scheme 1A. In details, DF-PEG-DF and Glycol chitosan were dissolved in DI water to form the solution, respectively. Then the hydrogel was constructed via mixing the DF-PEG-DF solution within the glycol chitosan solution at a ratio of 4:1 (v/v). The viscosity and stiffness of hydrogel could be optimized by adjusting the relative concentration of the crosslinker (DF-PEG-DF) or the polymer (chitosan-NH2). To prepare the DDMH, DTX/PLGA NPs and DOX were previously dispersed in DF-PEG-DF solution, and the as-synthesized Fe3O4@DF-PEG-DF nanoparticles (Fe3O4@DF-PEG-DF NPs) were previously added into the Glycol chitosan solution because the amino group on the surface of Fe3O4@DF-PEG-DF NPs can reacted with phenyl aldehyde group of chitosan-NH2, which would result in a uniform distribution of Fe3O4@DF-PEG-DF NPs in DDMH (Scheme 1B). The SEM image of DDMH treated by lyophilisation (Scheme 1C) revealed that the DDMH had a 3D porous structure with pore size ranging between 100 ∼ 200 µm. Such porous
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structure can allow the encapsulated DOX and DTX to be released from the hydrogel via diffusion. The hysteresis loop (Scheme 1D) suggested that both the Fe3O4@DFPEG-DF NPs and DDMH showed superparamagnetic behaviors and after encapsulated in hydrogel, the saturation magnetization of Fe3O4@DF-PEG-DF NPs decreased from 73.3 emu/g to 55.8 emu/g. The superparamagnetic property of DDMH will play an essential role for the induced heating properties of DDMH under the alternative magnetic field (AMF).
The shear strain-dependent viscoelastic properties and the hydrogel kineties of DDMH were evaluated by the measurement of the storage modulus (G’) and loss modulus (G’’) in terms of time. The stiffness and gelation time of DDMH were shown in Figure 1A. The gelation time for DDMH was about 180 s and the elastic modulus (G’) was 1.2 kPa at room temperature. Moreover, after being diluted, the gelation time decreased and the elastic modulus (G’) changed from 1.2 kPa to 290 kPa. In the whole frequency range tested (1-100 Hz, Figure 1C), G’ was consistently greater than G”, indicating the DDMH was stable and behaved like a viscoelastic solid. In order to further evaluate the elastic response of DDMH, G’ and G” of DDMH on strain amplitude sweep (γ=0.01%-10%) at a fixed angular frequency of 1 rad s-1 was measured. As shown in Figure 1D, the G’ curve intersected the G” curve at the strain of 2%, which means that when the strain was larger than this critical value, the G’ was lower than G”, which indicated that the sol-to-gel transition and the collapse of DDMH. And this could be contributed to the dynamic bonds between amino group and phenyl aldehyde group.
3.2. Injectable and self-healing behaviour of DDMH. The strain-induced damage and selfhealing behaviour of DDMH were measured through the continuous oscillatory strain step change between 1% and 300% under a constant frequency of 1 Hz. The structure destruction of DDMH was induced by applying a 300% strain for 120 s and then another 120 s was used for the recovery of the hydrogel via decreasing the strain from 300% to 1%. Figure 2A shows
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the damage of DDMH after the application of a higher shear strain of 300% and the selfhealing process at the lower strain of 1%. The gel-to-sol transition occurred when a higher dynamic strain decreased the elastic modulus from 1.2 kPa to 15 Pa due to the dissociation of Schiff base linkages.40 And the sol-to-gel transition appeared when a lower strain was applied because under a lower dynamic strain, the elastic modulus (G’) quickly recovered to the initial value of 1.2 kPa from ≈15 Pa. The gel-to-sol and sol-to-gel transition indicated that DDMH could restore its original microstructure after a higher strain-induced structural damage, namely, DDMH had the self-healing behaviour, which was obviously confirmed the adaptability properties of DDMH at room temperature after been injected above the glass beads (Figure 2C). The DDMH flowed gradually through the gap of the beads and finally formed complete hydrogel which encapsulated glass beads at the bottom of tube 2 h later.
According to the results of the rheological recovery property (Figure 2A), the self-healing DDMH was sol state under higher shear strain and returned to gel state when the stress was removed. Therefore, DDMH could be used as injectable gel materials for local or intravitreal injection. To conform this, the DDMH was loaded into syringe and extruded through 26guage needle. The smooth letters “HYDROGEL” shown in Figure 2B revealed that the hydrogel was injectable. When the DDMH in the syringe was compressed, the pressure, which acted like a high dynamic strain, made the hydrogel “flow” like liquid to pass through the needle due to the dissociation of Schiff base linkages mentioned above. Then the extruded hydrogel returned to gel state for the removing of pressure. The self-healing and injectable behaviours of DDMH indicate that it may be used deliver drugs and implant to body with a minimally invasive strategy.
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3.3. In vitro 3D culture and biocompatibility. Hydrogel is a promising material acting as extracellular matrix mimics for 3D culture of cells in the application of tissue engineering,41,42 drug discovery43 and cancer therapy.44,45 As we know, most of cells will lose their functionality and viability after being isolated in hydrogel. However, self-healing hydrogels are the potential materials to overcome the obstacle because of its dynamic behaviour under different intensity of shear strain. Therefore, mouse L929 fibroblastic cells which have been widely used as an in vitro model to investigate the cytotoxicity and biocompatibility of biomaterials, were used for 3D culture in Fe3O4@DF-PEG-DF loaded hydrogel (magnetic hydrogel) to test its cytocompatibility. 46 The results were shown in Figure 3. The spatial distribution of cells in magnetic hydrogel was shown in Figure 3C, showing that the encapsulated cells in hydrogel was spherical and could not spread over. Fluorescent confocal microscopy images (Figure 3A1, A2, B1, B2) of L929 cells which were stained with Calcein AM/propidium iodide showed that the L929 cells could tolerate the 3D encapsulation both in blank hydrogel and magnetic hydrogel. Meanwhile, the cell viability (Figure 3D) was as high as 99.8% and 99.3% after 48 h culture for blank hydrogel and magnetic hydrogel, respectively. The results indicated that the fibroblast cells could live well in the self-healing magnetic hydrogel network. We suggested that the self-healing magnetic hydrogel could not only act as extracellular matrix mimics which provided a microenvironment for cell living, but also deliver nutrition to the cells.
3.4. In vitro drug release assay. The accumulative release profiles of chemotherapeutic agents DOX and DTX from the DOX-loaded hydrogel (DOX Hydrogel), DTX-loaded hydrogel (DTX Hydrogel), DOX- and DTX-loaded hydrogel (DOX + DTX Hydrogel), DOXand DTX/PLGA NPs-loaded hydrogel (DOX + DTX/PLGA NPs Hydrogel) and DDMH are shown in Figure 4. The amount of DOX and DTX are detected by UV-vis-NIR
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spectrophotometer and HPLC, respectively. It is obviously that the release of both DOX and DTX from the different hydrogels are time-dependent and exhibit sustained-release properties (Figure 4A, B).
Compared with DTX Hydrogel, the sustain release of DTX from
DTX/PLGA Hydrogel was slow and there was no DTX during the first 24 h (Figure 4B). A more interesting result was that the release rate of DOX and DTX was mutual promotion (Figure 4C) in both DOX + DTX Hydrogel and DOX + DTX/PLGA Hydrogel, which meant that the release speed of one drug was increased by the releasing process of another one compared with the isolated release process. Within 30 days, the accumulative release amount of DOX released from DOX + DTX Hydrogel increased from 48.9% to 67.6% compared to DOX Hydrogel due to the existence of DTX, and in the same way, the amount of DTX increase from 78.2% to 83.7% due to the influence of DOX. For DOX is hydrophilic 47 and DTX is hydrophobic, 48 we speculated that they will repel with each other due to the hydrophilic and hydrophobic interactions, which then will speed up the diffusion process in hydrogel matrix. This indicated that the co-delivery modal could affect and/or control the release of the drug from the DDMH for the interaction between two drug molecules. To conform the phenomenon, DTX/PLGA NPs were used to replace the free DTX in hydrogel (Figure 4D) for a multi-stage controlling release of DTX. Because DTX is encapsulated with PLGA, the release of DOX and DTX from the hydrogel was asynchronous, which was verified by the fact that there was no DTX in the system of DOX + DTX/PLGA NPs Hydrogel in the first 24 h. Moreover, the releasing speed of DOX from DOX + DTX/PLGA Hydrogel was same with DOX Hydrogel at first but became faster after DTX released form DTX/PLGA NPs. Meanwhile, the releasing rate of DTX from DOX + DTX/PLGA Hydrogel was obviously larger than that from DTX/PLGA Hydrogel (Figure 4B, D), which further confirmed the mutual interactions between the releasing behaviours of DOX and DTX. For the multi-stage release properties of DTX from PLGA spheres in hydrogel and the
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interactions between DOX and DTX molecular, the asynchronous releasing of DOX and DTX may be a potential way to overcome the multi-drug resistance for chemotherapy of cancer.
The VSM image of DDMH (Scheme 1D) have shown that DDMH was superparamagnetic and had high saturation magnetization. Therefore, the induced heating property were measured under a AMF with 282 kHz frequency (Figure 5A). The temperature rises of DDMH under a high power intensity of 19.99 kA/m, which induced a temperature rise of ≈27.6℃ within 300 s, were found to be much higher than 7.7℃ under low power intensity (13.33 kA/m). Meanwhile, there was a little difference between different volume of DDMH (Figure 5B), which indicated that an effective temperature change could be obtained for small dose in vivo. The induction heating behaviour of DDMH indicated that as-synthesized DDMH was thermo-sensitive and the AMF could be to control the temperature of DDMH, then to control the release of drugs in DDMH. Figure 5C and 5D showed the releasing behaviours of DOX and DTX from DDMH with or without AMF, respectively. AMF treatment could speed up the release of DTX form the DDMH, while there was no significant difference for DOX before and after the heating. We assume that the AMF induced heating had no influence on the diffusion of drug molecules in the three-dimension loose structure of DDMH. But the diffusion process of DTX from the PLGA nanoparticles could be accelerated by heating because the structure of PLGA nanoparticle changed under a higher temperature environment. Previous studies have shown that the release of drug from the PLGA nanoparticles could be controlled by an outer temperature switch like AMF-triggered heating. Many researches have tried to reveal the principle of temperature induced release in PLGA,49 - 51
one of the reasons is that a higher temperature would increase the diffusion rate of drug
molecules in PLGA and speed up the biodegradation rate of PLGA itself. The difference
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AMF-trigger releasing properties between DOX and DTX in DDMH make it possible for us to adjust the chemotherapy process for cancer in the co-delivery system.
3.5. In vitro cytotoxicity assays. To demonstrate the synergistic therapeutic effect of DDMH, the in vitro antitumor efficacy of free drugs and drug-loaded hydrogels against the triple negative breast cancer cell line (MDA-MB-231) were measured by CCK-8 assay. In this work, CI50 (inhibitory concentration to produce 50% cell death) is used to evaluate the synergistic effect of co-delivery, whose definition is:
ܫܥହ =
(ܥܫହ ) (ܥܫହ ) + (ܥܫହ )௦ (ܥܫହ )௦
Figure 6 and Table 1 showed the viabilities of cells and the value of IC50 and CI50 of free drugs in co-delivery system after incubation for 12, 24 and 36 h. the value of IC50 and CI50 was calculated using the software of SPSS (IBM Statistics SPSS 19). It is obvious that the value of CI50 is smaller than 1 when the incubation time was larger than 24 h., which indicates that the synergistic therapy between DOX and DTX to triple negative breast cancer cell. Meanwhile, the lower CI50 value of 36 h compared to that of 24 h revealed that the control release of DTX from DTX/PLGA NPs could continuously assisted with DOX to kill the cancer cells. The noticed variation of the CI50 values in different treatment time periods (12, 24 and 36 h) may indicate the combined drug effect of docetaxel and doxorubicin on the cell line is time dependent and complicated.
We further evaluated the viability of MDA-MB-231 cells treated with different drug-loaded hydrogels. And it could be observed from the Figure 7 that all the drug-loaded hydrogels
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showed time-dependent cell proliferation inhibition behaviours and the co-delivery of DOX and DTX/PLGA NPs leaded to enhanced cell proliferation inhibition. Meanwhile, in the first 24 h, the antitumor therapeutic effect of DOX was more effective compared with DTX/PLGA NPs because there was few DTX released from DTX/PLGA NPs hydrogel due to the multistage release process of PLGA nanoparticles. The synergistic effect of co-delivery system was obvious for the cell viability is lower than 19.6% after 48 h incubation compared with DOX Hydrogel (43.6%) and DTX/PLGA NPs Hydrogel (54.1%). In our research, the thermo-sensitive property of DDMH provided an AMF-trigger control release of drugs for more efficient antitumor effect. After 48 h incubation, the result showed that a lowest cell viability of cells (5.6%) was obtained. The synergistic therapeutic effect indicated DDMH was a much promising co-delivery system for delivering therapeutic agents for efficient triple negative breast cancer treatment.
3.6. Toxicology evaluation of DDMH. To reveal potential toxicity of DDMH on treated mice, the blood biochemistry and haematology analysis of DDMH to Balb/c female mice was investigated over 12 weeks (Figure 8). The Balb/c female mice injected with DDMH (200 µL) were killed for blood collection at 1, 3 and 12 weeks post injection, respectively. The levels of importance markers revealing liver functions, including alkaline phosphatase, alanine aminotransferase and albumin concentration were all within normal ranges,
52
indicating that the treatment of DDMH had no prominent hepatic toxicity (Figure 8A). The urea levels in the blood, which is a one of key indicators for the kidney functions, were also within the normal range (Figure 8B). Furthermore, platelets, mean corpuscular haemoglobin concentration, mean haemoglobin, mean corpuscular volume, white blood cells, haemoglobin and red blood cells were selected for the haematological assessment (Figure 8C-I), and all the indicators showed that the mice treated with DDMH were normal compared to the control
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group. And this was further confirmed by the histology essay of major organs (skin, heart, liver, spleen, lung and kidney) which were collected from both the untreated mice and DDMH-injected mice at 1, 3, and 12 weeks after injection (Figure S5 (Supporting Information)). Therefore, we concluded that DDMH showed no obvious toxicity, which indicated a broad applications of DDMH as nanomedicine.
3.6. In vivo anti-cancer efficacy. In vivo anti-tumor effect was conducted in MDA-MB-231 tumor-bearing nude mice. Two groups of mice were intratumoral (i.t.) injected with Saline and DDMH. After 24 h injection, the mice were exposed to an AMF for 10 min with an intensity of 19.99 kA/m (the frequency is 282 kHz) and the real-time temperature of the tumor site were recorded with an IR thermal camera (Figure 9A). After the 10 min of AMF exposure, the tumor injected with DDMH had a maximum temperature of 48.0 °C (Figure 9A1), however, the saline-treated tumor under the same AMF exposure only produced a temperature change of 1.3 °C (Figure 9A2), which was insufficient to trigger the drug release in co-delivery system. The chemotherapy efficacies in MDA-MB-231 tumor-bearing mice were evaluated by measuring the tumor sizes of each group at certain day after injection (Figure 9B). The results showed that the growth of MDA-MB-231 tumors was mildly inhibited by DOX or by DTX/PLGA NPs compared with the rapid growth in Saline group. Although DOX and DTX are efficient chemotherapeutic agents against to MDA-MB-231 tumor, multiple and high dosages are required in order to obtain a satisfied antitumor effect, which will result in high toxicity and produce MDR effect.53 In the current study, the mice which were administrated only with a single dose of DOX showed that it was not sufficient to inhibit the tumor growth in vivo. However, DTX/PLGA NPs Hydrogel group showed a more effective inhibition for the multi-stage release of DTX from DOX/PLGA NPs hydrogel. The co-delivery DOX + DTX/PLGA NPs Hydrogel group revealed significantly an enhanced
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antitumor activity compared with single DOX Hydrogel group or DTX/PLGA NPs Hydrogel group, which agreed with the result in vitro (Figure 7), indicating the synergistic antitumor effect of the co-delivery system. As was shown in Figure 5 C and D, an outer AMF could speed up the releasing process of drugs from hydrogel. Therefore, in vivo antitumor effect was evaluated by exposed the DDMH-injected mice under a AMF for 10 min, and the result demonstrated that an excellent antitumor activity was obtained after 4 days treatment compared with the mice in DOX + DTX/PLGA NPs Hydrogel group which were not triggered by AMF.
The change of body weight change is a key indicator for evaluation of systemic toxicity. As shown in Figure 9C, the average body weights of saline, DOX hydrogel, DTX/PLGA NPs hydrogel, DOX + DTX/PLGA NPs hydrogel and DDMH-treated mice exhibited a slow but continuous increase during the 14 days treatment, which might be partly attributed to the normal tumor growth and low toxicity of different hydrogels. Obvious weight loss after 2 days was observed in mice treated different hydrogels compared to the saline-treated mice. We speculated that mice were scared during the i.t. injection process but it returned to normal level after 4 days. With the significant antitumor efficiency and the low drug-related toxicity, our DDMH system is promising multi-agent co-delivery system delivering drugs for synergistic chemotherapy of cancer.
4. CONCLUSION
In summary, we had successfully developed a dual-drug-loaded magnetic hydrogel (DDMH) for synergistic chemotherapy of triple negative breast cancer. The as-prepared DDMH not only had good biocompatibility but also exhibited unique self-healing, injectable and thermo-
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sensitive properties. Both in vitro and in vivo antitumor assays demonstrated that a remarkably improved synergistic antitumor activity to triple negative breast cancer cell line was obtained compared to DOX- or DTX/PLGA NPs-loaded hydrogels. Moreover, the asynchronous control release and AMF-trigger control release of DOX and DTX in codelivery system was proved to have more efficient antitumor effect of cancer chemotherapy. Therefore, we concluded that DDMH was a promising multi-agent co-delivery system delivering drugs for synergistic chemotherapy in the cancer treatment field.
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FIGURES
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Scheme 1. Preparation of DDMH. (A) Glycol chitosan crosslinking with DF-PEG-DF through the benzaldehydes at both terminals to form injectable and self-healing hydrogel; (B) Schematic illustration of the morphology and microstructure of the DDMH; (C) The SEM image of DDMH; (D) Hysteresis loop analysis of Fe3O4@DF-PEG-DF NPs and DDMH.
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Figure 1. Rheological properties of DDMH. (A, B) The gelation time, the storage moduli G’ and loss moduli G” of DDMH at 25℃; (C) The modulus (G’ and G”) from frequency sweep; (D) The modulus (G’ and G”) from strain amplitude sweep of the hydrogels.
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Figure 2. (A) The damage-self-healing property of DDMH revealed via the continuous step strain (from 1% to 300% and then 300% to 1%) evaluation at 37 °C; (B) The DDMH can get through a 26-gauge needle smoothly (with an inner diameter of 260 µm) without blockage. (C) The self-healing behaviours and adaptability of DDMH after being injected above the glass beeds.
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Figure 3. Three-dimensional culture of L929 cell lines in DDMH. Confocal images of L929 cells stained by Calcein-AM and PI after incubated with blank hydrogels (A1, A2) and DDMH (B1, B2) for 48 h. Z-Scans show the spatial distribution of cells; (C) Microscopy image of L929 cells in DDMH; (C) 4 weeks and (D) The total number of cells after incubated with vacant hydrogels and DDMH for 48 h;
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Figure 4. Cumulative release profiles of DOX and DTX at 37℃ from different kinds of drugloaded hydrogel formulation; (A) DOX release profile; (B) DTX release profile; (C, D) The synergistic releasing behaviours between DOX and DTX from different hydrogel formulation. Each point was an average value of three individual measurements.
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Figure 5. (A, B) Hyperthermia study showing the time, concentration and intensity of AMFdependent temperature rise of DDMH, the frequency of AMF was 282 kHz. The inner photos were real infrared thermal images of DDMH under AMF; (C, D) Controlling cumulative release of DOX and DTX from DDMH with or without AMF, respectively. Each point was an average value of three individual measurements.
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Figure 6.
Cell viabilities of MDA-MB-231 cells after treatment with combined
chemotherapy of DOX and DTX/PLGA NPs. Values are mean ± SD; n = 5.
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Figure 7. Cell viabilities of MDA-MB-231 cells after been treated with single chemotherapy and combined chemotherapy. Cell viability of MDA-MB-231 Cell viability was determined using Cell Counting Kid 8 (CCK-8) assay. Values are mean ± SD; n = 5. (* p < 0.05, ** p < 0.001.)
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Figure 8. Blood biochemistry and haematology data of Balb/c female mice treated with 200 µL DDMH at 1, 3 and 12 weeks: (A) ALT, ASP and ALP levels in the blood at 1, 3 and 12 weeks after DDMH injection. (B) The changes of BUN. (C-I) The changes of platelets (C), mean corpuscular haemoglobin concentration (D), mean haemoglobin (E), mean corpuscular volume (F), haemoglobin (G), red blood cells (H) and white blood cells (I) from control group and DDMH-treated group. Values are mean ± SD; n = 3.
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Figure 9. A) Infrared thermal image for tumor-bearing mice exposed to an AMF (19.99 kA/m) for 10 min at post-injection of saline and DDMH (100 µL). A1 and A2) The tumor site temperature during the explosion. B) Tumor growth curves with different treatments (Saline group, DOX group, DTX/PLGA NPs group, DOX + DTX/PLGA NPs group, DDMH + AMF group. Data presented the mean ± standard deviation of five mice. C) Average body weights of the mice of different groups after different treatment. Values are mean ± SD; n = 5. (* p < 0.05, ** p < 0.001.)
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TABLE
Table 1. In vitro cytotoxicity and combination index (CI) of drug formulations against MDAMB-231 cells for 12, 24, 36 h incubation time. Incubation Time (h)
IC50 (DOX/DTX) (g/L)
CI50
12
1.024/3.629
1.518
24
0.093/0.339
0.138
36
0.066/0.234
0.092
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ASSOCIATED CONTENT
Supporting information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04424.
1
H NMR spectrum of DF-PEG-DF. SEM and TEM images of Fe3O4 MNPs, Fe3O4@DF-
PEG-DF NPs and DTX/PLGA NPs. SEM image of Fe3O4-loaded hydrogel and Fe3O4@DFPEG-DF-loaded hydrogel. Histology evaluation of the major organs.
AUTHOR INFORMATION
Corresponding Author *E-mail:
[email protected]. Author Contributions # Zhao LY and Wei Y designed the study and wrote the protocol. Xie WS completed most part of experiments and performed statistical analysis, and then prepared the manuscript. Gao Q, Guo ZH, Wang D and Gao F participated in parts of the experiments. Wang XM contributed on the manuscript revision. All authors read and approved the final manuscript. Notes The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
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This study was supported by grants of the National Natural Science Foundation of China (No. 81671829).
REFERENCES
1
Ferlay J.; Soerjomataram I.; Dikshit R.; Rser S.; Mathers C.; Rebelo M.; Parkin D. M.; Forman D.; Bray F. Cancer Incidence and Mortality Worldwide: Sources, Methods and Major Patterns in GLOBOCAN 2012. Int. J. Cancer 2015, 136, E359-E386. 2 Edwards B. K.; Noone A. M.; Mariotto A. B.; Simard E. P.; Boscoe F. P.; Henley S. J.; DCM A. J.; Cho H.; Anderson R. N.; Kohler B. A.; Eheman C. R.; Ward E. M. Annual Report to the Nation On the Status of Cancer, 1975-2010, Featuring Prevalence of Comorbidity and Impact on Survival Among Persons with Lung, Colorectal, Breast, or Prostate Cancer. Cancer 2014, 120, 1290-1314. 3 Schnipper L. E.; Davidson N. E.; Wollins D. S.; Tyne C.; Blayney D. W.; Blum D.; Dicker A. P.; Ganz P. A.; Hoverman J. R.; Langdon R.; Lyman G. H.; Meropol N. J.; Mulvey T.; Newcomer L.; Peppercorn J.; Polite B.; Raghavan D.; Rossi G.; Saltz L.; Schrag D.; Smith T. J.; Yu P. P.; Hudis C. A.; Schilsky R. L. American Society of Clinical Oncology Statement: a Conceptual Framework to Assess the Value of Cancer Treatment Options. J. Clin Oncol 2015, 33, 2563-2577. 4 Pelaz B.; Alexiou C.; Alvarez-Puebla R. A.; Alves F.; Andrews A. M.; Ashraf S.; Balogh L. P.; Ballerini L.; Bestetti A.; Brendel C.; Bosi S.; Carril M.; Chan W. C. W.; Chen C.; Chen X.; Chen X.; Cheng Z.; Cui D.; Du J.; Dullin C.; Escudero A.; Feliu N.; Gao M.; George M.; Gogotsi Y.; Grünweller A.; Gu Z.; Halas N. J.; Hampp N.; Hartmann R. K.; Hersam M. C.; Hunziker P.; Jian J.; Jiang X.; Jungebluth P.; Kadhiresan P.; Kataoka K.; Khademhosseini A.; Kopeček J.; Kotov N. A.; Krug H. F.; Lee D. S.; Lehr C.; Leong K. W.; Liang X.; Ling L. M.; Liz-Marzán L. M.; Ma X.; Macchiarini P.; Meng H.; Möhwald H.; Mulvaney P.; Nel A. E.; Nie S.; Nordlander P.; Okano T.; Oliveira J.; Park T. H.; Penner R. M.; Prato M.; Puntes V.; Rotello V. M.; Samarakoon A.; Schaak R. E.; Shen Y.; Sjöqvist S.; Skirtach A. G.; Soliman M. G.; Stevens M. M.; Sung H.; Tang B. Z.; Tietze R.; Udugama B. N.; VanEpps J. S.; Weil T.; Weiss P. S.; Willner I.; Wu Y.; Yang L.; Yue Z.; Zhang Q.; Zhang Q.; Zhang X.; Zhao Y.; Zhou X.; Parak W. J. Diverse Applications of Nanomedicine. ACS Nano 2017, 11, 2313-2381. 5 Heldin C. H.; Rubin K.; Pietras K.; Pietras K.; Ostman A. High Interstitial Fluid Pressure-An Obstacle in Cancer Therapy. Nat. Rev. Cancer 2004, 4, 806-813. 6 Wicki A.; Witzigmann D.; Balasubramanian V.; Huwyler J. Nanomedicine in Cancer Therapy: Challenges, Opportunities, and Clinical Applications. J. Controlled Release 2015, 200, 138-157. 7 Hu C. Y.; Bailey C. E.; You Y. N.; Skibber J. M.; Rodriguez-Bigas M. A.; Feig B. M.; Chang G. J. Time Trend Analysis of Primary Tumor Resection for Stage IV Colorectal Cancer: Less Surgery, Improved Survival. JAMA surgery 2015, 150, 245-251. 8 Antoni M. H.; Jacobs J. M.; Bouchard L. C.; Lechner S. C.; Jutagir D. R.; Gudenkauf L. M.; Blomberg B. B.; Gluck S.; Carver C. S. Post-Surgical Depressive Symptoms and Long-Term Survival in Non-Metastatic Breast Cancer Patients at 11-Year Follow-Up. Gen Hosp Psychiatry 2017, 44, 16-21. 9 Estanqueiro M.; Amaral M. H.; Conceição J.; Lobo J. M. S. Nanotechnological Carriers for Cancer Chemotherapy: the State of the Art. Colloids Surf., B 2015, 126, 631-648. 10 Iwamoto T. Clinical Application of Drug Delivery Systems in Cancer Chemotherapy: Review of the Efficacy and Side Effects of Approved Drugs. Biol. Pharm. Bull. 2013, 36, 715-718. 11 Liang X.; Gao J.; Jiang L.; Luo J.; Jing L.; Li X.; Jin Y.; Dai Z. F. Nanohybrid Liposomal Cerasomes with Good Physiological Stability and Rapid Temperature Responsiveness for High Intensity Focused Ultrasound Triggered Local Chemotherapy of Cancer. ACS nano 2015, 9, 1280-1293. 12 She W.; Li N.; Luo K.; Guo C.; Wang G.; Geng Y.; Gu Z. Dendronized Heparin−Doxorubicin Conjugate Based Nanoparticle as PH-Responsive Drug Delivery System for Cancer Therapy. Biomaterials 2013, 34, 2252-2264. 13 Alimohammadi Y H, Joo S W. PLGA-Based Nanoparticles as Cancer Drug Delivery Systems. Asian Pac. J. Cancer Prev. 2014, 15, 517-535.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Allen T. M.; Cullis P. R. Liposomal Drug Delivery Systems: from Concept to Clinical Applications. Adv. Drug Delivery Rev. 2013, 65, 36-48. 15 McConville C.; Tawari P.; Wang W. Hot Melt Extruded and Injection Moulded Disulfiram-Loaded PLGA Millirods for the Treatment of Glioblastoma Multiforme via Stereotactic Injection. Int. J. Pharm. 2015, 494, 7382. 16 Weaver C. L.; LaRosa J. M.; Luo X.; Luo X.; Cui X. T. Electrically controlled Drug Delivery From Graphene Oxide Nanocomposite Films. ACS nano 2014, 8, 1834-1843. 17 Singh N. K.; Lee D. S. In Situ Gelling PH-and Temperature-Sensitive Biodegradable Block Copolymer Hydrogels for Drug Delivery. J. Controlled Release 2014, 193, 214-227. 18 Merino S.; Martín C.; Kostarelos K.; Prato M.; Vazquez E. Nanocomposite Hydrogels: 3D Polymer– Nanoparticle Synergies for On-Demand Drug Delivery. ACS nano 2015, 9, 4686-4697. 19 Colby A. H.; Colson Y. L.; Grinstaff M. W. Microscopy and Tunable Resistive Pulse Sensing Characterization of the Swelling of PH-Responsive, Polymeric Expansile Nanoparticles. Nanoscale 2013, 5, 3496-3504. 20 Huang F.; You M.; Chen T.; Zhu G.; Liang H.; Tan W. Self-Assembled Hybrid Nanoparticles for Targeted CoDelivery of Two Drugs Into Cancer Cells. Chem. Comm. 2014, 50, 3103-3105. 21 Guo Y.; He W.; Yang S.; Zhao D.; Li Z.; Luan Y. Co-Delivery of Docetaxel and Verapamil by Reduction-Sensitive PEG-PLGA-SS-DTX Conjugate Micelles to Reverse the Multi-Drug Resistance of Breast Cancer. Colloids Surf., B 2017, 151, 119-127. 22 Noh I.; Kim H. O.; Choi J.; Choi Y.; Lee D. K,; Huh Y.; Haam S. Co-Delivery of Paclitaxel and Gemcitabine via CD44-Targeting Nanocarriers as a Prodrug with Synergistic Antitumor Activity Against Human Biliary Cancer. Biomaterials 2015, 53, 763-774. 23 He C.; Lu K.; Liu D.; Lin W. Nanoscale Metal–Organic Frameworks for the Co-Delivery of Cisplatin and Pooled Sirnas to Enhance Therapeutic Efficacy in Drug-Resistant Ovarian Cancer Cells. J. Am. Chem. Soc. 2014, 136, 5181-5184. 24 Kemp J. A.; Shim M. S.; Heo C. Y.; Kwon Y. J. “Combo” Nanomedicine: Co-Delivery of Multi-Modal Therapeutics for Efficient, Targeted, and Safe Cancer Therapy. Adv. Drug Del. Rev. 2016, 98, 3-18. 25 Chen Y.; Zhang W.; Huang Y.; Gao F.; Sha X.; Fang X. Pluronic-Based Functional Polymeric Mixed Micelles for Co-Delivery of Doxorubicin and Paclitaxel to Multidrug Resistant Tumor. Int. J. Pharm. 2015, 488, 44-58. 26 Zhu H.; Wang Y.; Hussain A.; Shen Y.; Guo S. Nanodiamond Mediated Co-delivery of Doxorubicin and Malaridine to Maximize Synergistic Anti-Tumor Effects on Multi-Drug Resistant MCF-7/ADR Cells. J. Mater. Chem. B 2017, 5, 3531-3540. 27 Wang H.; Zhao Y.; Wu Y.; Hu Y.; Nan K.; Nie G.; Chen H. Enhanced Anti-Tumor Efficacy by Co-Delivery of Doxorubicin and Paclitaxel with Amphiphilic Methoxy PEG-PLGA Copolymer Nanoparticles. Biomaterials 2011, 32, 8281-8290. 28 Lv S.; Tang Z.; Li M.; Lin J.; Song W.; Liu H.; Huang Y.; Zhang Y.; Chen X. Co-Delivery of Doxorubicin and Paclitaxel by PEG-Polypeptide Nanovehicle for the Treatment of Non-Small Cell Lung Cancer. Biomaterials 2014, 35, 6118-6129. 29 Usami T.; Kato K.; Taniguchi T.; Abe A.; Nomura H.; Yamamoto A.; Matoda M.; Okamoto S.; Kondo E.; Omatsu K.; Kawamata Y.; Takeshima N. Recurrence Patterns of Advanced Ovarian, Fallopian Tube, and Peritoneal Cancers after Complete Cytoreduction During Interval Debulking Surgery. Int. J. Gynecol. Cancer 2014, 24,991-996. 30 Hayashi K.; Ono K.; Suzuki H.; Sawada M.; Moriya M.; Sakamoto W.; Yogo T. High-Frequency, Magnetic-FieldResponsive Drug Release from Magnetic Nanoparticle/Organic Hybrid Based on Hyperthermic Effect. ACS Appl. Mater. Interfaces 2010, 2, 1903-1911. 31 Loi S.; Sirtaine N.; Piette F.; Salgado R.; Viale G.; Eenoo F. V.; Ronas G.; Francis P.; Crown J. P. A.; Hitre E.; Azambuja E. D.; Qiunaux E.; Leo A. D.; Michiels S.; Piccart M. J.; Sotiriou C. Prognostic and Predictive Value of Tumor-Infiltrating Lymphocytes in a Phase III Randomized Adjuvant Breast Cancer Trial in Node-Positive Breast Cancer Comparing the Addition of Docetaxel to Doxorubicin with Doxorubicin-Based Chemotherapy: BIG 0298. J. Clin. Oncol. 2013, 31, 860-867. 32 Blay J. Y.; Leahy M. G.; Nguyen B. B.; Patel S. R.; Hohenherger P.; Santoro A.; Staddon A. P.; Penel N.; Piperno-Neumann S.; Hendifar A.; Lardelli P.; Nieto A.; Alfaro V.; Chawla S. P. Randomised Phase III Trial of Trabectedin Versus Doxorubicin-Based Chemotherapy as First-Line Therapy in Translocation-Related Sarcomas. Eur. J. Cancer 2014, 50, 1137-1147. 33 Loriot Y.; Massard C.; Gross-Goupil M.; Palma M. D.; Escudier B.; Bossi A.; Chauchereau A.; Fizazi K. The Interval from the Last Cycle of Docetaxel-Based Chemotherapy to Progression is Associated with the Efficacy of Subsequent Docetaxel in Patients with Prostate Cancer. Eur. J. Cancer 2010, 46, 1770-1772.
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Ploussard G.; Terry S.; Maillé P.; Alloy Y.; Sirab N.; Kheuang L.; Soyeux P.; Nicolaiew N.; Coppolani E.; Paule B.; Salomon L.; Culine S.; Buttyan R.; Vacherot F.; Taille A. D. L. Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy. Cancer Res. 2010, 70, 92539264. 35 Swift L. P.; Rephaeli A.; Nudelman A.; Phllips D. R.; Cutts S. M. Doxorubicin-DNA Adducts Induce a NonTopoisomerase II–Mediated form of Cell Death. Cancer Res. 2006, 66, 4863-4871. 36 Yang F.; Teves S. S.; Kemp C. J.; Henikoff S. Doxorubicin, DNA Torsion, and Chromatin Dynamics. Biochimi. Biophys. Acta 2014, 1845, 84-89. 37 Yuan J.; Liu J.; Song Q.; Wang D. Xie W. S.; Yan H.; Zhou J.; Wei Y.; Sun X.; Zhao L. Y. Photoinduced Mild Hyperthermia and Synergistic Chemotherapy by One-Pot-Synthesized Docetaxel-Loaded Poly (lactic-co-glycolic acid)/Polypyrrole Nanocomposites. ACS appl. mater. Interfaces 2016, 8, 24445-24454. 38 Chou T. Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies. Pharmacol. Rev. 2006, 58, 621-681 39 Latham A. H.; Williams M. E. Versatile Routes toward Functional, Water-Soluble Nanoparticles via Trifluoroethylester− PEG− Thiol Ligands. Langmuir 2006, 22, 4319-4326. 40 Ding F.; Wu S.; Wang S.; Xiong Y.; Li Y. Li B.; Deng H.; Du Y.; Xiao Y.; Shi X. A Dynamic and Self-Crosslinked Polysaccharide Hydrogel with Autonomous Self-Healing Ability. Soft Matter 2015, 11, 3971-3976. 41 Tibbitt M. W.; Anseth K. S. Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture. Biotechnol. Bioeng. 2009, 103, 655-663. 42 Nicodemus G. D.; Bryant S. J. Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications. Tissue Eng., Part B 2008, 14, 149-165. 43 Nyga A.; Cheema U.; Loizidou M. 3D tumour Models: Novel in Vitro Approaches to Cancer Studies. J. Cell Comm. Signaling 2011, 5, 239-248. 44 Thoma C. R.; Zimmermann M.; Agarkova I.; Kelm J. M.; Krek W. 3D Cell Culture Systems Modeling Tumor Growth Determinants in Cancer Target Discovery. Adv. Drug Delivery Rev. 2014, 69, 29-41. 45 Seliktar D. Designing cell-Compatible hydrogels for biomedical applications. Science 2012, 336, 1124-1128. 46 Watarai A.; Schirmer L.; Thönes S.; Freudenberg U.; Werner C.; Simon J. C.; Anderegg U. TGFβ Functionalized Starpeg-Heparin Hydrogels Modulate Human Dermal Fibroblast Growth and Differentiation. Acta Biomater. 2015, 25, 65-75. 47 Xu J.; Zhao Q.; Jin Y.; Qiu L. High Loading of Hydrophilic/Hydrophobic Doxorubicin into Polyphosphazene Polymersome for Breast Cancer Therapy. Nanomedicine 2014, 10, 349-358. 48 Hwang H. Y.; Kim I. S.; Kwon I. C.; Kim Y. Tumor Targetability and Antitumor Effect of Docetaxel-Loaded Hydrophobically Modified Glycol Chitosan Nanoparticles. J. Controlled Release 2008, 128, 23-31. 49 Loo J. S. C.; Ooi C. P.; Boey F. Y. C. Degradation of Poly (lactide-co-glycolide)(PLGA) and Poly (L-lactide)(PLLA) by Electron Beam Radiation. Biomaterials 2005, 26, 1359-1367. 50 Grayson A. C. R.; Cima M. J.; Langer R. Size and Temperature Effects on Poly (lactic-co-glycolic acid) Degradation and Microreservoir Device Performance. Biomaterials 2005, 26, 2137-2145. 51 Zolnik B. S.; Leary P. E.; Burgess D. J. Elevated Temperature Accelerated Release Testing of PLGA Microspheres. J. Controlled Release 2006, 112, 293-300. 52 Yang K.; Wan J.; Zhang S.; Zhang Y.; Lee S.; Liu Z. In Vivo Pharmacokinetics, Long-Term Biodistribution, and Toxicology of Pegylated Graphene in Mice. ACS Nano 2011, 5, 516–522. 53 Holmes F. A.; Jones S. E.; O’shaughnessy J.; Vukelja S.; Grorge T.; Savin M.; Richards D.; Glaspy J.; Meza L.; Cohen G.; Dhami M.’ Budman D. R.; Hackett J.; Brassard M.; Yang B. B.; Liang B. C. Comparable Efficacy and Safety Profiles of Once-Per-Cycle Pegfilgrastim and Daily Injection Filgrastim in Chemotherapy-Induced Neutropenia: a Multicenter Dose-Finding Study in Women with Breast Cancer. Ann. Oncol. 2002, 13, 903-909.
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GRAPHICAL ABSTRACT
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