Ying Yanga,
Lin Zhub,
Feng Xiaa,
Baoyou Gonga,
Anjian Xiea,
Shikuo Lia,
Fangzhi Huanga,
Shaohua Wanga,
Yuhua Shen*a and
David T. Weaver*b
aSchool of Chemistry and Chemical Engineering, Anhui University, Hefei 230601, P. R. China. E-mail: s_yuhua@163.com
bInstitute of Health Sciences, Anhui University, Hefei 230601, P. R. China. E-mail: David.weaver.t@gmail.com
First published on 12th January 2017
In this study, a novel drug-loaded inorganic nanoparticle–biomolecule hybrid hydrogel shell containing fluorouracil (5-FU)/reduced graphene oxide (rGO)/Brassica chinensis extract (Bce) on tumor cells was firstly constructed through a facile one-step method. The Bce stems from a natural green vegetable for improving the bio-compatibility of 5-FU/rGO/Bce hydrogel and presents multi-functions in this system. As both a reducing and a cross-linking agent for GO, Bce is beneficial for the fast formation of a rGO/Bce hybrid hydrogel at body temperature (37 °C) before laser irradiation, as well as being an excellent photo-sensitizer for inducing the generation of cytotoxic singlet oxygen (1O2) to kill tumor cells by means of laser radiation. The rGO in the hybrid hydrogel possesses photothermal characteristic for the hyperthermia antitumor. After loading anticancer drugs, the formed 5-FU/rGO/Bce hybrid hydrogel shells encapsulated on the tumor cells in situ exhibit three competitive advantages, i.e., retaining the high concentration of drugs around tumor cells to enhance the localized antitumor effect, reducing the side effects by hindering drugs from migrating to normal tissue, and displaying effectively synergetic chemo/photo-thermal/photodynamic therapies. This study provides a new strategy to exploit the multi-functions of hybrid hydrogel materials for effective biomedical applications.
In recent years, some highly effective anti-cancer therapies with few or minimal undesired toxic effects have been studied, such as photo-thermal therapy (PTT) and photodynamic therapy (PDT). PTT5–8 with little damage from the near infrared (NIR) laser to the human body and a short treatment time of a matter of minutes could reduce the pain experienced by sufferers. However, PTT could hardly be used alone for cancer treatment due to the limited lethality. On the other hand, PDT9–13 exhibits good selectivity and can be repeated, which does not affect the function of the hematopoietic and immune system. Nevertheless, improper selection of the photo-sensitizer can also cause adverse reactions of light allergy, such as a local skin rash or blisters.14–17 According to the above proposed obstacles, we aim to construct a suitable hydrogel carrier containing a photo-thermal agent and a photo-sensitizer combined with chemotherapeutic drugs on tumor cells for an effective and localized therapy, which proceeds in the following three main aspects.
Firstly, graphene oxide (GO)-based hydrogel,18,19 a kind of new material developed in recent years, has excellent mechanical properties and biocompatibility. The edge of the GO nanosheet is rich in carboxyl, hydroxyl and epoxy groups, which are very easy to directly combine with hydrophilic drugs. At the same time, the sp2 conjugate region of the GO surface layer can also provide a platform for the loading of hydrophobic drugs,20,21 so the GO-based hydrogel can be used as a commendable drug carrier. In addition, GO and its chemical derivatives possess a certain photo-thermal conversion ability and also can be used in the PTT.22–24
Then, the direct synthesis of multi-function biological medicine by using natural products instead of noxious chemicals, is a current research trend in the biological medicine field. For this purpose, our daily vegetables (or their extracts) are a great choice. For example, our group has used pepper extract to prepare Se and Ag nanoparticles;25 some research groups26,27 use fresh alfalfa, lemon grass and citronella geranium leaves to synthesize gold and silver nanoparticles through a one-step method. Our group has also previously used amaranth extract (ARE) to prepare a GO-based hydrogel.28 In this work, Brassica chinensis, a traditional green vegetable grown in China and Southeast Asia, has a long history of cultivation and mature cultivation techniques, and is not limited by the seasons compared to ARE, which was chosen as the novel raw material of cross-linking and reducing agent to prepare GO-based hydrogel through a one-step and facile method. In addition, Bce possesses extremely plentiful biomolecules containing chlorophyll, amino acids, sugars, vitamin C (Vc), folic acid and enzymes,29–31 which could transform the GO into reduced graphene oxide (rGO) along with the formation of a rGO/Bce hydrogel network through the main driving forces of redox, hydrogen bonding, π–π interaction, or electrostatic interaction between GO and Bce.28,32 In addition, Bce also can be used as a photo-sensitizer due to the enriched chlorophyll, leaf protein and reduced amino acids encapsulated in the hydrogel, which induced the release of singlet oxygen (1O2) under laser irradiation for the realization of PDT. This idea and practice has not been reported before.
Finally, recent research shows that a synthetic cell shell33 that simulates other biological natural shell-like cocoons, diatoms, egg, etc.,34,35 has important significance and broad application prospects in the aspect of cell protection, storage, transportation and treatment. Inspired by these, to synthesize a smart drug loaded shell on the surface of the specific cancer cell is a novel idea for the application of tumor therapy with a high efficiency and low toxicity.
In our work, we report a convenient and green route for the three-dimensional self-assembly of GO nanosheets with Bce and 5-FU chosen as a model anticancer drug, and a novel multifunctional 5-FU/rGO/Bce hybrid hydrogel shell formed in situ on the cancer cells. In this hybrid hydrogel shell, the high concentration of drugs could be retained around tumor cells through the fast gelation of 5-FU/rGO/Bce at body temperature (37 °C) to enhance the effect of localized antitumor through synergetic chemo/photo-thermal/photodynamic therapy. This study provides a new strategy to exploit multi-functions of hybrid hydrogel materials for effective biomedical applications.
UV-Vis spectra of samples were obtained on a U-3900 UV spectrophotometer (Hitachi, Japan) in the range of 200–1000 nm. Scanning Electron Microscopy (SEM) images were taken by using an S4800 scanning electron microscope operated at 5 kV. X-ray Diffraction (XRD) patterns were performed on an XD-3 X-ray diffractometer using a Cu-Kα radiation source (λ = 1.5418 Å). The atomic force microscopy (AFM) images were obtained by using a Veeco Multimode 8 scanning probe microscope in the tapping mode. Fourier transform infrared (FT-IR) spectra were acquired using a NEXUS-870 spectrophotometer (Thermo Fisher, USA, frequency range from 4000 to 500 cm−1) with a KBr pellet. Electron spin resonance spectroscopy (ESR, BRUKER E500) was carried out with microwave frequency of 9.78 GHz, modulation frequency of 100 kHz, microwave power of 10 mW, modulation amplitude of 1 G, time constant of 40 milliseconds, field sweep of 100 G and center field of 3480 mT. Fluorescence images were recorded on a DMI3000B inverted fluorescence microscope (Leica, German). IR thermal imaging was performed with an IR thermal camera (Fluke, Everett, WA). Raman spectra of the samples were obtained using a Jobin Yvon Lab Ram HR 800 equipped with a 532 nm laser (Renishaw, UK). The OD values of MTT assay were measured by a RT-2100C spectro-photometric micro-plate reader (Rayto, Shenzhen, P. R. China). The chromatograms were obtained by high performance liquid chromatography (HPLC) equipment that consisted of a Waters Delta 600 solvent delivery system, a Waters 717 plus Autosampler, a Waters 2998 Photodiode array detector, a Waters 600 controller and a data acquisition/processing computer with Empower™ software (Waters, Milford, MA), and the analyses were performed in a SinoChrom ODS-BP 5 PoChrom OD (4.6 × 250 mm) at ambient temperature with a mobile phase of phosphoric acid/methanol (98:2, v/v) at a flow rate of 1.0 mL min−1. The injection volume was 20 μL. On-line UV spectra were recorded in the range of 190–600 nm and the λ 242 nm trace was used for the calculation of peak areas for all the samples.
In addition, the generation of 1O2 could be also tested by an ESR method. β-DM (5.2 mg), TEMP (249.5 mg) and Bce (1.5 mg) were mixed with a tricine–NaOH buffer solution (50 mL, 50 mmol L−1, pH 8.0) to the prepare sample. For the specific experimental methods, refer to our previous work.28 The corresponding ESR spectra of Bce were recorded after illumination with a 650 nm red laser for 30 min. The experiments were independently repeated at least three times.
1 mL of DI water or rGO/Bce dispersion was added in a small bottle and then irradiated with an 808 nm NIR laser at a power density of 1.0 W cm−2 at regular time intervals (1 min). The infrared (IR)-thermal images and photo-thermal heating curves of the above samples were collected by a Fluke Ti32 thermal IR camera for investigating the photo-thermal effect of the rGO/Bce hybrid hydrogel.
(1) |
In addition, the effect of different volume ratios of 5-FU (2.0 mg mL−1) loaded in GO to Bce (V5-FU/GO:VBce = 0.5:1, 1:1, 1.5:1, and 2:1) on the formation of the 5-FU/rGO/Bce hydrogel at room temperature (25 °C) and body temperature (37 °C) were studied.
(2) |
To study the cellular uptake ability of 5-FU/rGO/Bce, HeLa cells (5 × 104 per well) were seeded in 6-well plates and cultured in medium for 24 h (37 °C, 5% CO2). After complete adhesion, the cells were washed twice with serum-free medium. Then, 3 mL of fresh serum-free medium containing 1 mL mixture of 5-FU/rGO/Bce sol and fluorescein isothiocyanate (FITC)–dextran (1 mg mL−1) was added and incubated at 37 °C for 1 h. The remaining steps are similar to our previous work.28,37
The fast gelation of rGO/Bce may be ascribed to the strong interaction between GO and Bce, including amide bond, π–π stacking, hydrogen bonding, electrostatic attraction, etc., accompanied by the reduction of GO to rGO contributed to by a vitamin such as Vc or reducing amino acids in Bce, which was confirmed by following the XRD patterns, FTIR spectra, Raman spectra, HPLC chromatograms and so on.
Fig. 2A gives the XRD patterns of GO (a), Bce (b) and rGO/Bce (c) hybrid hydrogel, respectively. As shown in Fig. 2A-a, pure GO reveals a sharp (002) diffraction peak at 2θ of 11.33°, relating to a layer-to-layer stacking distance of 7.82 Å.38 Fig. 2A-b shows the XRD pattern of the Bce, demonstrating that bio-molecules in Bce are amorphous. The broad (002) diffraction peak around 27.24° that appears in Fig. 2A-c indicates that the interlayer distance of rGO/Bce decreased to 3.34 Å, suggesting an ample reduction of GO by Bce.
The FT-IR spectra of GO, Bce, and rGO/Bce hybrid hydrogel are shown in Fig. 2B-a–c, respectively. In the case of GO (Fig. 2B-a), an intense and broad band is observed at about 3200–3400 cm−1 due to the O–H stretching vibrations arising from hydroxyl groups in GO. The absorption at 1725 cm−1 is the characteristic band of CO groups in carbonyl and carboxyl moieties. The three characteristic absorbance peaks of Bce at 1636, 1520 and 1246 cm−1 in Fig. 2B-b are assigned to the C–O stretching vibration of –NHCO– (amide I), the N–H bending vibration of –NH2 (amide II) and the C–N bending vibration of –NHCO– (amide III),25,39 indicating the existence of proteins in Bce. The bands at 1079 cm−1 and 1400 cm−1 could be attributed to the C–O–C group of chlorophyll, reducing vitamin (Vc) and polypeptide amino acid, etc. After GO was mixed with Bce, the FTIR spectrum of the obtained rGO/Bce hybrid hydrogel from Fig. 2B-c reveals the disappearance of the –COOH group of GO at 1725 cm−1, and the broadening of the amide I band at ∼1636 cm−1. Meantime, the peak intensity of the acetylated amino group –NHCO– (amide II and amide III) decreased. The above peak changes disclose that there were strong interactions between the polar groups of GO and Bce, which act as crosslink agents for the reduction of GO and the formation of the rGO/Bce hybrid hydrogel.
The Raman spectra of GO and the rGO/Bce hybrid hydrogel are shown in Fig. 2C, which could further demonstrate the reduction of GO and successful fabrication of the rGO/Bce hydrogel. Two strong peaks at 1355 cm−1 and 1593 cm−1 assigned to the typical D and G bands of GO40 are shown in Fig. 2C-a. However, the corresponding two bands for the rGO/Bce hydrogel from Fig. 2C-b display a small red-shift to 1347 cm−1 and a small blue-shift to 1600 cm−1.41 The spectral shifts could be ascribed to the disturbance of the GO structure caused by physical or chemical interactions between the epoxy, hydroxyl, and carboxyl moieties of GO and polar groups of Bce, such as the amino, carboxyl, acylamino and alkoxy groups. At the same time, the ID/IG value of the rGO/Bce hybrid hydrogel decreased from 0.81 to 0.78 in comparison with that of the GO (Fig. S1†). These results can be attributed to the processes of the relatively green and facile chemical reduction of GO compared with solvothermal method42 and the strong bonding effect of GO and Bce.
We also examined the electrochemical behavior of Bce to determine the role of Bce as a reducing agent. Fig. 2D shows a typical cyclic voltammogram of the Bce, which was recorded at a scan rate of 0.02 V s−1 in the potential window between −1.50 and 0.90 V. It can be seen clearly from the black line in Fig. 2D that there is no redox activity at the Pt electrode in pure water in the potential range. However, an oxidation peak at the potential of about 0.50 V in the Bce can be seen,43 indicating the occurrence of an oxidation reaction for Bce, which possibly arose from the reducing activity of the hydrosulfuryl, hydroxyl and amino groups from vitamins such as Vc or reducing amino acids in the Bce. According to our investigations, Vc, as a strong reducing agent, has a similar electrochemical behavior to that of Bce (from a previous report).44 Thus, we hypothesized that Vc may play a major role in the formation of a rGO/Bce hydrogel.
To further confirm the above speculation, a HPLC method was chosen to measure the changes of Vc content in Bce before and after the addition of GO. Fig. 2E shows an HPLC chromatogram of the Vc standard detected by the absorbance at 242 nm.45 The retention time of the Vc was 7.3 min. A typical chromatogram of the Vc in Bce is also observed in Fig. 2F-a with a peak area of 11219185 mAU s. We can also see from Fig. 2F-a that the contents of other compositions in Bce are relatively much less than that of Vc. After the addition of a GO dispersion, the peak of Vc shown in Fig. 2F-b is greatly decreased compared with the peak area of 552091 mAU s. Therefore, the reduction effect of Bce to GO is also demonstrated by the decrease in peak area of Vc detected at 242 nm. We can conclude that Vc played an important role in the formation of the rGO/Bce hydrogel.
By SEM observation (Fig. 3), we found that there were some obvious differences between the morphologies of GO and the rGO/Bce hybrid hydrogel. Fig. 3A shows a wrinkled paper-like morphology of GO. In addition, the AFM image of GO nano-sheets on the mica wafer is presented in Fig. S2-A,† showing that GO nanosheets have an irregular shape with the plane sizes in the range from tens to thousands of nanometers and an average thickness of ∼1.3 nm (ref. 25) (Fig. S2-B†), which is the characteristic thickness of single-layer GO nano-sheets. Compared with pure GO, a 3-D porous network structure is found in the rGO/Bce hybrid hydrogel with an average pore size of ∼30 μm (Fig. 3B and the corresponding inset (a)), which is attributed to the thicker layers of stacked GO nano-sheets through strong interactions between the GO and Bce.
Fig. 3 SEM images of (A) GO and (B) the rGO/Bce hydrogel. Inset (a) in (B) is the corresponding partial high magnification version. |
The 1O2 generation that may be induced by the GO, Bce and rGO/Bce systems is very important for PDT. DPBF reacts with 1O2 irreversibly and undergoes a 1,4-cycloaddition reaction that is detected as a decrease in the absorption intensity of the DPBF at 410 nm.25 The UV-Vis spectrum changes of the mixed GO/DPBF system are shown in Fig. 4B; it is seen that the DPBF absorption peak at 410 nm has no significant decline with laser irradiation (650 nm) for 20 s. However, we clearly observe that the absorption peak intensity of the Bce/DPBF (Fig. 4C) or rGO/Bce/DPBF (Fig. 4D) system at about 410 nm is rapidly weakened, demonstrating that 1O2 was generated quickly and DPBF was consumed fast, suggesting that Bce/DPBF or rGO/Bce/DPBF used to determine the production of 1O2 is available and the efficient generation of 1O2 in the photosensitized process is mainly due to the contribution of Bce and not of GO. To further confirm the results of the DPBF experiments, ESR spin trapping spectroscopy for Bce was measured. 1O2 can be measured by ESR because it can oxidize TEMP to form the stable N-oxyl radical TEMPO. The characteristic symmetrical triplet of the paramagnetic TEMPO ESR spectrum47 is obviously observed from Fig. 4E. The signal of 1O2 production further indicates that Bce can be used in PDT. These results suggest that the rGO/Bce hybrid hydrogel can act as an effective photodynamic agent for the release of 1O2 to kill tumor cells.
We also investigated the potential use of rGO/Bce as a photothermal agent. The red curve of rGO/Bce in Fig. 4F presents a rapid temperature increase during the initial irradiation for 4 min and a slow temperature variation from 4 to 10 min. The total temperature increase of rGO/Bce hydrogel is up to 17 °C within 11 min, whereas the water temperature increases by only 3 °C under the same laser exposure. The IR-thermal images of water and the rGO/Bce hybrid hydrogel exposed to the laser for 11 min are shown in insets (a) and (b) in Fig. 4F, which demonstrate the temperatures at 28 °C and 42 °C, respectively. These results further confirm that the rGO/Bce hybrid hydrogel has a good photo-thermal effect and could serve as a synergistic platform for PDT/PTT with an efficient therapy.
The initial volume ratio of V5-FU/GO:VBce was chosen to further investigate the influence of 5-FU loading on the stability of the hybrid sol or hydrogel. Fig. 6 reveals the state of the 5-FU/rGO/Bce hybrid sol or hydrogel with different initial V5-FU/GO:VBce at room temperature (25 °C) and simulative body temperature (37 °C). Obviously, the optimum V5-FU/GO:VBce is 1.5:1 (corresponding to the mass concentration ratio of 6:17) for the formation of a 5-FU/rGO/Bce hybrid hydrogel at 37 °C, which is similar to the results of Fig. 1. Comparing Fig. 1B or C with Fig. 6A or B, we can also find out that the loading of 5-FU to rGO/Bce has an inconspicuous effect on the stability of the 5-FU/rGO/Bce sol or hydrogel. Thus, we can directly inject the 5-FU/rGO/Bce sol with V5-FU/GO:VBce in the range 0.5:1 to 1.5:1 at room temperature into the lesion site to form a hydrogel through minimally invasive surgery at body temperature.
Scheme 1 Schematic diagrams of the fabrication process for the 5-FU/rGO/Bce hybrid hydrogel and the antitumor mechanism of the 5-FU/rGO/Bce hybrid hydrogel shell formed on the HeLa cells. |
The formation of the hybrid hydrogel on the surface of HeLa cells in situ was investigated by optical microscope photos and fluorescence images (Fig. 7). The microscopic images of the HeLa cells in the absence or presence of 5-FU/rGO/Bce hybrid hydrogel encapsulation are shown in Fig. 7A and B. It can be seen from Fig. 7B that the surface of the HeLa cells is covered with a thin and semitransparent shell compared with those in Fig. 7A. Fig. 7C shows a fluorescence microscopy image of HeLa cells encapsulated with hydrogel shells stained by Hoechst 33342, and Fig. 7D presents the merged image of Fig. 7B and C. Thus, a firm and apparent hydrogel shell (indicated by red arrows) on the HeLa cells can be seen more clearly from Fig. 7D. The results indicate that the hydrogel shell can be easily and successfully formed on the HeLa cells in situ.
Fluorescence images of HeLa cells under different conditions are shown in Fig. 9, which are used to further illustrate the bio-compatibility and antitumor activity of the hybrid hydrogel. For HeLa cells incubated with nothing or rGO/Bce without irradiation, cell deaths are both difficult to observe (essentially no red in Fig. 9A2 and B2) due to the favorable biocompatibility of the rGO/Bce hydrogel. Fig. 9C shows cells incubated with the 5-FU/rGO/Bce without irradiation; some cells appear stained by PI (red fluorescence in Fig. 9C2), indicating the toxicity of the released 5-FU from 5-FU/rGO/Bce, in agreement with the MTT results. More dead cells stained by PI (faint red fluorescence signals in Fig. 9D2) could be seen from the rGO/Bce experimental group with irradiation, which reveals that Bce and rGO irradiated by laser can induce the generation of 1O2 and hyperthermia to kill HeLa cells together. Among Fig. 9C–E, the strongest red fluorescence images of HeLa cells incubated with 5-FU/rGO/Bce in the presence of laser irradiation can be observed in Fig. 9E2 and E3. Such a fact demonstrates that the antitumor effect of the 5-FU/rGO/Bce hybrid hydrogel under irradiation is much higher than that without irradiation, suggesting multimodel synergetic therapies, such as CT, PDT and PTT due to the coexistence of 5-FU, rGO and Bce, are superior to a single one for cancer treatment. As a result, the hydrogel system may be used in localized collaborative treatment for cancer.
FITC–dextran notation can monitor samples in and out of the cell membrane, so we use FITC–dextran to label the 5-FU/rGO/Bce hybrid hydrogel. Fig. 10A shows the image of FITC–dextran with HeLa cells, there is almost no fluorescence within the cells. However, after incubation with the FITC–dextran-labeled 5-FU/rGO/Bce hybrid hydrogel, an intense green fluorescence is clearly visible in the entire cell with laser excitation (Fig. 10B). This contrast result could be explained by the multi-interactions (hydrogen bonding, van der Waals forces, hydrophobic interactions, etc.) between the 5-FU/rGO/Bce hybrid hydrogel and sugar chains or phospholipid molecules on the cell membrane, such that the 5-FU/rGO/Bce hybrid hydrogel can induce the destabilization of the lipid bilayer membrane and then act as a trans-membrane carrier to increase the internalization of 5-FU/rGO/Bce in the cells, which will be favorable for a dense distribution of drugs, including 5-FU, photosensitizers and photothermal agents, on lesions and hinder their migration to normal tissue at the same time, and thereby strengthen the therapeutic effect.
Fig. 10 Fluorescence microscopy images of (A) FITC–dextran with HeLa cells and (B) FITC–dextran labeled 5-FU/rGO/Bce with HeLa cells. The scale bars are 50 μm. |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra25834d |
This journal is © The Royal Society of Chemistry 2017 |