Qing Wu,
Zhaoqi Wang,
Haixia Zhang,
Rongrong Zhu,
Shilong Wang* and
Qigang Wang*
Department of Chemistry, School of Life Science and Technology, Tongji University, Shanghai 200092, China. E-mail: wsl@tongji.edu.cn; wangqg66@tongji.edu.cn
First published on 25th May 2016
This communication describes a new strategy to fabricate a nanogel layer around magnetic nanoparticles by surface free-radical polymerization triggered by the cascade reaction of urate oxidase and horseradish peroxidase, which showed high loading capacity, pH-responsive drug release and low cytotoxicity.
Herein, we develop a new strategy to fabricate multifunctional core–shell nanogels by the surface free-radical polymerization initiated by the cascade reaction of urate oxidase (UOx) and horseradish peroxidase (HRP). UOx is a peroxisomal enzyme which can catalyze the oxidation of uric acid to allantoin.33 However, most microbial UOx present unsatisfactory enzyme activity and stability under physiological conditions.33–36 Therefore, the catalytic activity of UOx would have a great degree reduction after the surface-initiated polymerization of nanogels, which will benefit the biomedical applications of the enzyme-initiated system because of no cytotoxicity from the further radical after polymerization. Magnetic nanoparticles (MNPS) are selected as a very intriguing candidate of inorganic cores due to their easily separation characterization, while the intrinsic magnetic property can benefit the potential applications, such as magnetic-targeting, thermotherapy, and imaging.37–41 The surface UOx around MNPs catalyzes the oxidative transformation of uric acid to allantoin with concomitant hydrogen peroxide (H2O2), which reacts with ACAC via HRP catalysis to generate ACAC radicals at the interface to initiate the polymerization and gelation. The functional monomer methacrylic acid is selected to provide pH-responsive property within gel network. The pH-responsive hybrid nanogels exhibit not only a high drug loading capacity but also a pH-controllable drug releasing.
The proposed preparation of the multifunctional hybrid nanogels, is shown in Scheme 1. The magnetic Fe3O4 nanoparticles (MNPs) were synthesized according to the literature.42 And the average diameter of the obtained MNPs was approximate 40 nm. The surface of the obtained MNPs was first amino-functionalized by treatment with 3-aminopropyltriethoxysilane (APTES) to yield MNPs–APTES. Subsequently, the MNPs–APTES was further functionalized by reacting with succinic anhydride in DMF to achieve carboxylic functionalized nanoparticles MNPs–COOH. The zeta potential of the Fe3O4 nanoparticles with different surface modification was displayed in Fig. S2 (in ESI†). The carboxyl modified MNPs was then activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS). After wash, the acquired magnetite nanoparticles were combined with excess enzyme UOx and HRP solution (molar ratio of UOx:
HRP = 1
:
1) to covalently attach the UOx and HRP simultaneously to the carboxyl-modified MNPs. Afterwards, the as-prepared UOx/HRP-MNPs were redispersed in deionized water for further use after washed three times. The hydrogel was fabricated around the interface of UOx/HRP-MNPs with the addition of coating precursor solution, which was composed of PEGMA360 (4%, v/v), methacrylic acid (MAA, 2%, v/v), PEGDA250 (1%, v/v), ACAC (1%, v/v) and uric acid (0.02 M). The core–shell nanogels were acquired by magnetic separation from the solution after 12 h reaction and re-dispersed in water for further characterization.
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Scheme 1 The scheme of the preparation of the core–shell nanogels by UOx/HRP mediated hydrogelation. |
The surface gelation mechanism around MNPs is described as follows. The UOx surround the surface of the MNPs first catalyze oxidation of uric acid in the coating solution to generate of allantoic acid and H2O2, which interacts with the adjacent HRP on the MNPs and ACAC immediately in the bulk media to initiate the subsequent generation of ACAC radicals. Finally, the forming radicals around the UOx/HRP-MNPs can trigger the gelation of the hydrogel layer on the MNPs via the surface radical polymerization. The diffusion of ACAC radicals within the forming gel networks and short life time can prevent the likelihood of the polymerization in bulk phase. The easily deactivation of UOx during enzymatic reaction also reduce the bulk polymerization due to the low amount of radical. At the same time, the low residue activity of UOx after polymerization can reduce the cytotoxicity of the final nanogels in biological system that can interact with uric acid (Fig. S4 in ESI†). In consequence, the core–shell nanogels with high monodispersity were collected other than the aggregated bulk hydrogel.
As shown in Fig. 1, the morphology and size distribution of the collected core–shell nanogels were investigated using SEM, TEM and DLS measurements, respectively. The comparison of the SEM images of the air-dried Fe3O4 nanoparticles and the magnetic core–shell nanogels in Fig. 1a and b reveal that the nanogels are in a state of spherical shape and smooth surface with a diameter of 180 nm in dry state, while the magnetite Fe3O4 nanoparticles are coarse in surface. In addition, the nanogels exhibit more excellent monodispersity than the MNPs. The contrast of the surface appearance of MNPs and the core–shell structures indicates that the hydrogel layer has been successfully coated onto the surface of magnetite Fe3O4 nanoparticles. Furthermore, TEM images also demonstrate the differences of MNPs and nanogels in structure, as shown in Fig. 1d and S1 (in ESI†). The core–shell structure is quite explicit with a black core and a light contrast gray shell, while the initial MNPs have not this structure feature (Fig. S1 in ESI†), and hydrogel shell of the nanogels is about 10 nm in dry state according to the TEM image. The nanoparticles of both MNPs and nanogels were further characterized by size distribution (Fig. 1c). The average diameters of the MNPs and nanogels are approximate 80 nm and 200 nm, respectively, which is a little higher than that of SEM and TEM result in dried sample. The about 60 nm thickness in wet state also confirms the formation of apparent core–shell structure. Therefore, the pH responsive nanogels shells have been successfully coated on the MNPs through UOx/HRP initiated interfacial radical polymerization.
These core–shell nanogels can be employed as carriers for intracellular drug delivery in future cancer chemotherapy. To verify the feasibility, positively charged doxorubicin hydrochloride (DOX·HCl) was chosen as a model drug to evaluate the effect of the drug carrier systems. The DOX was loaded by mixing aqueous solutions of the core–shell nanogels and DOX·HCl in Tris·HCl buffer at pH 8.0, which was kept in dark place for 24 h. Free DOX was removed by magnetic separation and the loading content of DOX was determined by UV/Vis spectroscopy by measuring the absorbance of DOX at 480 nm. The negatively charged nanogels showed extremely high drug loading content and drug-loading efficiency (DLC = 88.1%, DLE = 66.0%). This high drug loading may be attributed to the interaction of negatively charged nanogels with the positively charged DOX.
To evaluate the feasibility of the DOX-loading nanogels for cancer therapy, the cytotoxicity of DOX-loaded nanogels, nanogels and free DOX toward SH-SY5Y cells were tested by MTT assay in Fig. 2. The SH-SY5Y cells were treated with varying concentrations of free and nanogels-loading DOX (0–20 μg mL−1) for 24 h and 48 h, respectively. At first, all the data showed that the blank core–shell nanogels without DOX exhibited >85% cell viability, suggesting unobvious cytotoxic effect of original nanogels on the cells. The cell viabilities with various form DOX and different concentration were investigated and shown in the Fig. 2. The cytotoxicity of DOX-loaded nanogels or pure DOX was observed by the significant growth inhibition of SH-SY5Y cells with the various drugs. The cell viability is about 28.4% when the 20 μg mL−1 nanogels-loading DOX was treated with SH-SY5Y cells for 48 h. The low cell viability suggested that DOX-loaded nanogels were efficiently taken up by the SH-SY5Y cells and released DOX in acidic microenvironment inside the cancer cells, which inhibited the cellular proliferation. At the same time, the cell viability is about 14.5% when the 20 μg mL−1 free DOX was treated with SH-SY5Y cells for 48 h. The lower cell viability of free DOX is reasonable because of the easily permeability of free DOX into cell. Our DOX-loaded nanogels are the better system than free one due to the selective uptake by tumour cells and pH responsive release to get better therapeutic efficiency.
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Fig. 2 Cytotoxicities of nanogels, DOX-loaded nanogels, and free DOX toward SH-SY5Y cells after incubation for 24 h (a) and 48 h (b). Error bar represented means of three independent experiments. |
The pH-triggered cumulative drug release of DOX from DOX-loaded nanogels in vitro was investigated at various pH values. Specifically, a certain amount of DOX-loaded nanogels including 600 μg DOX in dialysis bag was placed in PBS at different pH values of 4.5, 5.8, and 7.4. Fig. 3 shows the significant dependence of DOX release upon acidic pH values, and the release rate increased as the decreased pH value. As shown in Fig. 3, at endo-lysosomal acidity of pH 4.5, 55.5% DOX was released after 48 h incubation at 37 °C. In contrast, only 39.1% and 14.7% DOX were released at pH 5.8 and 7.4, respectively. The release rate of DOX enhanced with the decreased pH value, in the order of pH 4.5 > pH 5.8 > pH 7.4. The elevated released amount of DOX in low pH was attributed to the weakened electrostatic interaction between the positively charged DOX with negatively charged COO− groups of MAA in nanogel networks, which amount was inversely proportional to proton concentration according to the ionization equilibrium of methacrylic acid. Therefore, the pH-sensitive nanogels can possibly reduce the leakage of their embedded drug during transportation to the tumour regions while accelerate the drug release toward cancer cell in tumour region with more acidic microenvironment. The high drug loading content could benefit cancer therapy by the long-time cumulative release of DOX.
The cellular uptake and intracellular drug release of the DOX-loaded nanogels was furtherly confirmed by flow cytometry analysis (FACS) and Laser Confocal Scanning Microscopy (LCSM). The samples of SH-SY5Y cells were treated by DOX-loaded nanogels with 0.5–24 h incubation time. The quantitative characterization of various form drugs uptaked into the cells over time was done by FACS analyses. The flow cytometric histogram of the SH-SY5Y cells with DOX-loaded nanogels confirmed the high cellular uptake of the DOX-loaded nanogels, whose fluorescence intensity gradually increased with the increasing incubation time (Fig. S5 in ESI†). The cells nuclei were stained with DAPI for elevated contrast and clarity. As shown in Fig. 4, after 0.5 h incubation, tiny bright DOX fluorescence was observed in the cells, indicating the successful endocytosis of the DOX-loaded nanogels. Significantly, a stronger red fluorescence signal was found in the cell nuclei after 2 h incubation, demonstrating the fast cellular internalization of nanogels and speedy release of DOX which migrated into cell nuclei within 2 h. Similar to FACS analysis, the intensity of the red fluorescence was enhancing with an increase in the incubation time, suggesting that more DOX reached to cell nuclei. Meanwhile, considerable red fluorescence was also observed in the cytoplasm of cells.
Footnote |
† Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c6ra06331d |
This journal is © The Royal Society of Chemistry 2016 |