Shanshan Huanga,
Ping'an Ma*a,
Ziyong Chenga,
Bei Liua,
Xiaoran Denga,
Zhongxi Xieac,
Jun Lina and
Yanqiu Han*b
aState Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. E-mail: mapa675@ciac.ac.cn
bDepartment of Neurology, No. 2 Hospital, Jilin University, Changchun 130041, P. R. China. E-mail: yanqiu9@hotmail.com
cUniversity of Science and Technology of China, Hefei 230026, P. R. China
First published on 6th February 2017
Gd2O3:Yb3+,Er3+@nSiO2@mSiO2 (Gd@mSi) core/shell structure nanospheres were synthesized through a sol–gel method. Then biocompatible polysaccharide chitosan (CS) was grafted onto the surface of the nanoparticles to fabricate a pH responsive CS@Gd@mSi system. Furthermore, cancer targeting ligand folic acid (FA) was modified through the abundant amino groups on the chitosan polymer shell. The nanospheres with a Gd2O3:Yb3+,Er3+ core can be candidates for T1-weighted magnetic resonance imaging (MRI) contrast agents. The CS decorated nanocomposites showing good biocompatibility and red emission under 980 nm laser excitation can be potential candidates for bioimaging in vitro. FA modified nanospheres loaded with doxorubicin hydrochloride (DOX) show higher cytotoxicity for HeLa cells in vitro compared with those nanoparticles with chitosan shells only and pure DOX. The CS@Gd@mSi system can be a potential drug carrier with MRI, UCL, and finely controlled pH-dependent drug release properties.
Chitosan have been widely studied in biomedical applications based on its biocompatibility, biodegradability, bioactivity, antibacterial activity and the decoration of particles with specific ligands.16 Due to the abundant amino groups on the chains of the polymer, chitosan shows pH-sensitive features and has been used in drug delivery systems.17 The coating of chitosan was realized through various methods. MCM-41 type mesoporous silica nanoparticles (MSNs) decorated with chitosan have been synthesized based on the phosphoramidate chemistry18 and cross-linking reactions.19 Monodispersed chitosan–silica hollow nanospheres have been produced and investigated for delivering TNF-α to MCF-7 breast cancer cells under both in vitro and in vivo conditions.20 A biodegradable mesoporous chitosan–silica hybrid pH-responsive system has been synthesized by self-assembly of non-toxic F127 Pluronic non-ionic surfactant, biodegradable chitosan and silica source forming “NH2–Zn–DNR (daunorubicin)” coordination architecture.21
Gd2O3 nanoparticles have been widely studied for the existence of seven unpaired electrons in the 4f orbitals of Gd3+ ions can be used as effective imaging contrast agents in NMR imaging.22,23 Our group had synthesized composite composed of ordered mesoporous silica encapsulating Gd2O3:Eu3+ down-conversion luminescence nanoparticles24 and Gd2O3:Er3+ up-conversion luminescence nanoparticles25 with core shell structures and good biocompatibility as drug carriers. Though chitosan coated MSNs have been synthesized, upconversion luminescent Gd2O3:Yb3+,Er3+@nSiO2@mSiO2 decorated with chitosan have rarely been reported.
Based on our previous reports, we first synthesized Gd2O3:Yb3+,Er3+@nSiO2@mSiO2 nanoparticles with diameter of about 300 nm, then chitosan was coated onto the as-synthesized nanoparticles to realize the finely tuning the release of DOX response to the different pH environment. Folic acid was introduced to the chitosan due to the amino groups of the polymer as cancer targeting ligand. The anticancer drug doxorubicin (DOX) was loaded in the pores of the composite to investigate the pH-responsive controlled release behavior and cytotoxicity for HeLa cells. It was demonstrated that the DOX loaded CS@Gd@mSi and CS-FA@Gd@mSi system could be potential candidate for drug delivery in vitro.
The Gd2O3:Yb3+,Er3+@nSiO2@mSiO2 nanocomposite was prepared based on the previous report with some modification.24,25 In a typical procedure, the as-prepared Gd0.80Yb0.17Er0.03(OH)CO3 nanoparticles were treated with ethanol by ultrasonication for 30 min. Subsequently, the treated nanoparticles were separated by centrifugation, and then well dispersed in a mixture of ethanol (40 mL), deionized water (10 mL), and concentrated ammonia aqueous solution (28 wt%, 0.5 mL). TEOS (0.015 g) was then added dropwise to the solution. After stirred for 6 h, the products were separated by centrifugation and washed with ethanol and water, and then redispersed in a mixed solution containing cetyltrimethylammonium bromide (CTAB) (0.15 g), deionized water (40 mL), concentrated ammonia aqueous solution (28 wt%, 0.6 mL), and ethanol (30 mL). The resulting solution was stirred for 30 min. TEOS (0.2 g) was then added dropwise to the solution with stirring. After another stirring for 6 h, the products were collected and separated by centrifugation, washed with ethanol and water several times, and dried in air at 80 °C for 24 h and calcined at 550 °C for 6 h. Finally, the CTAB-removed product was denoted as Gd@mSi.
As the chitosan polymer own abundant amino groups, cancer cell targeting ligand folic acid was conjuncted onto the chitosan shell. 5 mg of CS@Gd@mSi were suspended in 2 mL dimethyl sulfoxide (DMSO) and sonicated for 10 min. 2 mg FA, 3.2 mg EDC and 2.2 mg NHS were dissolved in 2 mL DMSO and added into the above mixture and stirred overnight at room temperature. The materials were recovered by centrifugation and washed twice with DMSO and ethanol. The obtained sample was denoted as CS-FA@Gd@mSi.
For quantification of the cell uptake of the nanoparticles, HeLa cells were plated out in 6-well plates at a density of 2 × 105 cells per well and were allowed to attach and grow for 24 h. These cells were then incubated with CS@Gd@mSi (100 μg) and CS-FA@Gd@mSi (100 μg) at 37 °C for 1 h and 4 h. After washing with PBS three times, cells were lysed by cell lysis buffer. Gadolinium content in the cell lysis solution was measured by ICP-OES.
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Scheme 1 The fabrication process and pH responsive drug delivery of the multifunctional nanocomposites. |
The wide-angle XRD pattern of the Gd2O3:Yb3+,Er3+@nSiO2@mSiO2 after calcination was shown in Fig. 1. The broad band at 2θ = 22° can be attributed to the amorphous SiO2 (JCPDS 29-0085). The diffraction peaks at 2θ = 20.12° (211), 28.62° (222), 33.15° (400), 47.64° (440), 56.46° (622) can be indexed to phase for Gd2O3 (JCPDS no. 12-0797), which suggest that Gd2O3:Yb3+,Er3+ particles were well retained in the silica matrix.
As can be seen in Fig. 2, the as-synthesized Gd(OH)CO3:Yb3+,Er3+ consists of monodisperse nanospheres with a mean particle size of 200 nm. These particles with smooth surface show non-aggregation and narrow size distribution. Gd(OH)CO3:Yb3+,Er3+@nSiO2 with a solid silica layer was prepared through a modified Stöber procedure. The formation of mesoporous silica layer on the Gd(OH)CO3:Yb3+,Er3+@nSiO2 was performed by using CTAB as the organic template. After calcination at 550 °C for 6 h, the template removed samples was obtained and designated as Gd2O3:Yb3+,Er3+@nSiO2@mSiO2. The SEM images of the sample demonstrated that the particles owning uniform size and shape. After the coating of chitosan, the thin layer of chitosan can be seen in Fig. 2e, which confirms the successful introduction of the polymer. The EDX spectra in Fig. 2f confirmed the existence of Gd, Yb, Er and Si elements.
The TEM image of Gd2O3:Yb3+,Er3+@nSiO2@mSiO2 shows the core shell structure clearly because of the different electron penetrability between the solid Gd2O3:Yb3+,Er3+ core and mesoporous silica shell (Fig. 3). The Gd2O3:Yb3+,Er3+ cores are black spheres with mean diameter of about 150 nm, and the silica shell shows gray color with an average thickness of 50 nm. Ordered mesopore channels are found to be perpendicular to the sphere's surface. The chitosan thin layer can be seen clearly outside of the mesoporous silica. What's more, the mesopore channels can still be observed after the conjunction of the chitosan polymer. The DLS results are in agreement with the images shown in the SEM and TEM. The hydrodynamic diameter of CS@Gd@mSi varied from 317, 335, 342, 368 nm with pH (7.4, 6.0, 5.0, 4.0). It can be seen that the dimensions of the nanocomposites increased when the pH was lowered. The pH decrease of the PBS may lead to the protonation of the chitosan chains and induce the swelling of the nanocomposites.26
Fig. 4 demonstrates the N2 adsorption/desorption isotherm of Gd@mSi. The sample exhibited typical type IV isotherms, which confirm the presence of uniform mesoporous channels. The BET surface area of Gd@mSi is 441 m2 g−1, the pore volume is 0.29 cm3 g−1 and the average pore size of the sample is about 2.37 nm. This result indicates that the synthesized composites possess mesopore channels and space for the loading and entrance of drug molecules. According to the pore diameter and pore structure of the sample, we choose DOX which can be transported freely as the model drug. After the coating of chitosan, the BET surface and the pore volume of the sample decreased to 28.2 m2 g−1 and 0.03 cm3 g−1, respectively.
Fig. 5 displays the FTIR spectra of the prepared samples. All the samples displayed the stretching vibrations of Si–O–Si around 1084 cm−1 and 801 cm−1. CS@Gd@mSi show 2931, 2863 cm−1 ascribed to C–Hx stretching vibrations of alkyl groups after the coating of chitosan.15 This result indicated that chitosan had been grafted onto the surface of the particles through the GPTMS bridge. CS-FA@Gd@mSi shows peak at 1604 cm−1 ascribed to the N–H bending vibration of the CONH group, which confirmed the successful introduction of folic acid.27 The thermogravimetry analysis curves of the CS@Gd@mSi and CS-FA@Gd@mSi show 11%, 12% weight loss compared with the bare Gd@mSi nanoparticles, respectively (Fig. 6), which can be attributed to the degradation of chitosan molecules. These results can be further confirmed by the exothermic peaks in the range of 300–400 °C of the DSC curves for CS@Gd@mSi and CS-FA@Gd@mSi. While the Gd@mSi exhibited no exothermic peaks.
Fig. 7 shows the UCL spectrum of Gd@mSi under 980 nm laser excitation. The spectrum is composed of an intense band around 660 nm and some weak emission, which can be attributed to the 4F9/2–4I15/2 and 2H11/2/4S3/2–4I15/2 transitions of Er3+ ions,28 respectively. CS@Gd@mSi shows similar spectra with decreased intensities. The hydroxyl group possessing high-energy vibrational modes (2700–3600 cm−1) would quench the excited states of Er3+ ions by multiphonon relaxation and thus impact the upconversion processes.29–31 These results demonstrated that the chitosan coated particles can be potentially used for the cell imaging.
HeLa cells were incubated with the CS@Gd@mSi and CS-FA@Gd@mSi for 1 h and 4 h at 37 °C, and the luminescence images were taken with an external 980 nm laser as the excitation source. After rinsing with abundant PBS to remove surface-attached particles, the cells were investigated by UCLM (Fig. 8). The upconversion luminescence of the nanoparticles became brighter with increasing incubation time, which indicated that more and more nanoparticles were internalized into the cells. What's more, the UCL of CS-FA@Gd@mSi showed much brighter image compared with those nanoparticles without folic acid, which suggests that more CS-FA@Gd@mSi were internalized into HeLa cells.
To quantitatively evaluate the cell uptake of CS@Gd@mSi and CS-FA@Gd@mSi in HeLa cells, ICP-OES was used to measure the concentration of Gd3+ internalized by HeLa cells. The Gd3+ concentration for CS-FA@Gd@mSi in HeLa cells is measured to be 11.06, 13.92 μg mL−1 at 1 h and 4 h, respectively. While the values of Gd3+ concentrations for CS@Gd@mSi are 3.56 and 5.43 μg mL−1. Obviously, the uptake amount of CS-FA@Gd@mSi is almost 3 times as high as that of CS@Gd@mSi in HeLa cells. These results demonstrated that more and more nanoparticles were internalized into the HeLa cells with increasing time. What's more, it confirms that folic acid grafting is favorable for the cell uptake of the nanocomposites.32 It can be concluded that CS@Gd@mSi and CS-FA@Gd@mSi can be used as an excellent luminescence probe for cell imaging and monitoring the cell endocytosis process.
T1 relaxation time was measured in aqueous dispersions with different Gd3+ concentrations to evaluate the potential application of CS@Gd@mSi. As shown in Fig. 9, the longitudinal relaxivity (r1) was estimated to be 1.19 s−1 mM−1 from the slope of the relaxation rate (1/T1) as a function of Gd3+ concentration. The representative T1 weighted MR images of the CS@Gd@mSi suspensions clearly showed positive signal enhancement of the effect on T1-weighted sequences as the Gd3+ concentration increased, leading to brighter images. Compared with the previous reports, the longitudinal relaxivity (r1) value was in the same range with NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-PEG nanocomposites (0.83 s−1 mM−1).33 These results suggest that the as-prepared spheres could be employed as a potential T1 contrast agent.
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Fig. 9 (a) Relaxation rate R1 (1/T1) versus various molar concentrations of CS@Gd@mSi dispersions at room temperature. (b) T1 weighted images of various molar concentrations of Gd3+ in CS@Gd@mSi. |
The loading content of DOX into the composites was determined to be 8.0%. In vitro drug release behavior of the DOX loaded samples in PBS with various pH values (4.0, 5.0, 6.0 and 7.4) were investigated. It can be seen from Fig. 10 that the release of DOX from DOX-CS@Gd@mSi increased with decreasing pH value of the media. The left side image in Fig. 10 exhibits the magnification of the drug release in 8 h. In the first 0.5 h the drug released in PBS with lower pH show higher initial burst release. The DOX released in PBS (pH = 4.0) reached 62% within 4 h, which is much higher than others. At pH 7.4, about 19% of DOX was released out from within 72 h, while around 49%, 55% and 70% of DOX was released at pH 6.0, 5.0 and 4.0, respectively. When DOX-CS@Gd@mSi was immersed in acidic buffer solution, the chitosan polymer chains became positively charged due to the protonated amino groups, which lead to the swelling of the chitosan layers and opening of the mesopore of the particles.20 The loaded DOX molecules could be quickly released from the channels of the sample and diffuse into the aqueous solutions. While, at relatively high pH value (pH 7.4), the deprotonated polymer chains of chitosan condensed and form a shielding layer that covered the porous surface of the particles. The DOX was blocked in the mesopores of the sample and the release of the DOX molecules become slower compared with those in lower pH values. Therefore, the release of DOX can be finely tuned by the chitosan polymer layer.
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Fig. 10 Cumulative release profiles of DOX from DOX-CS@Gd@mSi systems in PBS with different pH values. |
To evaluate the cytotoxicity and biocompatibility of CS@Gd@mSi and CS-FA@Gd@mSi, the standard MTT cell assay was performed on L929 fibroblast cells. As shown in Fig. 11, more than 90% cell viability can be observed in the whole concentration range of 6.25–200 μg mL−1 after 24 h incubation with CS@Gd@mSi and CS-FA@Gd@mSi. It can be concluded that these nanoparticles have good biocompatibility.
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Fig. 11 The L929 fibroblast cell viability after incubating with different concentrations of CS@Gd@mSi and CS-FA@Gd@mSi nanoparticles for 24 h. |
The cytotoxicity effects of CS@Gd@mSi, CS-FA@Gd@mSi and DOX loaded samples were further tested in vitro with the MTT assay (Fig. 12). For pure CS@Gd@mSi and CS-FA@Gd@mSi nanoparticles, HeLa cells were not impaired obviously with a concentration as high as 625 μg mL−1 after the 24 h culture, indicating the good biocompatibility of the as-prepared nanocomposites. HeLa cells were incubated in a culture medium containing free DOX, DOX-CS@Gd@mSi and DOX-CS-FA@Gd@mSi for 24 h at different DOX concentrations. The inhibitory concentration values for DOX, DOX-CS@Gd@mSi and DOX-CS-FA@Gd@mSi showed an increasing inhibition against HeLa cells with an increased concentration. The DOX-CS@Gd@mSi exhibited similar cytotoxicity to that of free DOX, whereas DOX-CS-FA@Gd@mSi showed the highest cytotoxicity in these cases (Fig. 12). This may be attributed to the fact that FA modified nanoparticles can be taken up via receptor-mediated endocytosis by HeLa cells and DOX molecules were released inside to induce cell death. Compared with CS@Gd@mSi, folic modified particles can be taken up more into HeLa cells, which results in more DOX molecules released in the cells. The IC50 (the concentration of drug required to reduce cell growth by 50%) values found at 24 h for HeLa cells were 3.18, 6.87, and 5.15 μg mL−1 for DOX-CS-FA@Gd@mSi, DOX-CS@Gd@mSi and free DOX, respectively. The IC50 value of DOX-CS-FA@Gd@mSi was lower than the pure DOX. This might be attributed to the efficient cellular uptake of the FA modified sample. The higher IC50 value of DOX-CS@Gd@mSi than pure DOX may be ascribed to its comparable drug effect for inducing cancer cell death while the side effect of DOX was greatly abated due to the encapsulation of the nanocomposites.34 Based on these results, the CS-FA@Gd@mSi can potentially be used as a vector to store and transport anti-cancer drugs, target cancer cells and enhance the efficacy of anti-cancer drug delivery.
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Fig. 12 In vitro HeLa cell viabilities after incubation 24 h with CS@Gd@mSi, CS-FA@Gd@mSi, DOX-CS-FA@Gd@mSi, DOX-CS@Gd@mSi and pure DOX at different concentrations. |
This journal is © The Royal Society of Chemistry 2017 |