3.1 Synthesis of GO@Au-PEG/DOX
Figure 1 shows the synthesis scheme of GO@Au-PEG/DOX. During the synthesis of GO@Au, mild sodium citrate was selected as the reducing agent in the experiment, and gold nanoparticles were loaded on the surface of GO by hydrothermal method to form GO@Au nanocomposite. In GO@Au-PEG synthesis process, chloroformic acid p-nitrophenyl ester was used to activate the hydroxyl group on the GO surface, to get GO@Au of nitro phenyl acetic derivatives, then connect ethylenediamine chemical by ester bond on the GO@Au nanocomposites, to form the GO@Au-NH2, then introduced with hydrazine base HBA, add CHO-PEG2000, modified PEG by pH sensitive hydrazone bond on GO@Au nanocomposites. DOX is efficiently loaded onto the GO@Au-PEG surface through a strong π-π stacking.
3.2 Characterization of GO@Au-PEG
Figure 2a and b shows the TEM image of GO and GO@Au. It can be seen that GO is a typical stratified structure and Au is a sphere with a diameter of 10~20 nm. Au nanoparticles were uniformly distributed on the GO sheets, indicating that Au was successfully loaded on the GO@Au nanocomposite. Figure 2c shwos the UV spectrum of the GO and GO@Au. The the UV spectrum of GO@Au showed a strong absorption at 527 nm, showing that the gold nanoparticles are indeed loaded on the GO surface.
The FT-IR spectrum of GO, GO@Au, activated GO@Au, GO@Au-NH2, GO@Au-HBA and GO@Au-PEG are show in Figure 2d. GO shows an obvious absorption peak at 1710 cm-1, which is the absorption peak of -COOH carbonyl, and a strong absorption peak at 3400 cm-1, which is the characteristic peak of -OH. GO@Au shows an obvious absorption peak at 3400 cm-1, which means there remains a lot of -OH that can react with -OH on GO. When nitrophenyl was introduced and activated GO-Au was obtained, the -OH absorption peak was significantly weakened and N-O absorption peak appeared (1382 cm-1), indicating that nitrophenyl was successfully attached to GO@Au. Then, after ethylenediamine is conjugated on the activated GO@Au by an ester bond, the characteristic absorption peak of C-N appears at 1140 cm-1, the absorption peak of N-H bond appears at 1624 cm-1 and 3400 cm-1, and the absorption peak of N-O bond at 1382 cm-1 weakens, indicating the successful synthesis of GO@AU-NH2. When -NH2 is introduced into GO@Au, the HBA links to GO@Au through the reaction between -COOH and -NH2 to form amide bonds. Compared with GO@Au-NH2, GO@Au-HBA has absorption peaks at 1661 cm-1 and 1388 cm-1. Meanwhile, the absorption peaks of -NH2 at 1600 cm-1 and 992 cm-1 prove the existence of N-N bonds, indicating the successful synthesis of GO@Au-HBA. Finally, CHO-PEG2000 generates pH-sensitive hydrazone by reaction of aldehyde group with hydrazine group on HBA, and absorption peaks appear at 1716 cm-1 ,1241 cm-1 and 1600 cm-1, indicating successful preparation of GO@Au-PEG.
3.3 Characterization of GO@Au-PEG/DOX
As shown in Figure 3a, DOX has strong absorption peaks at 252 nm and 480 nm, GO@Au-PEG has a strong absorption peak at 527 nm, GO@Au-PEG/DOX has strong absorption peaks at 252, 480 and 527 nm. The mean Zeta potential of GO@Au-PEG/DOX was 35.5±2.6 mV, indicating that the Zeta potential of GO@Au-PEG/DOX was stable.
GO@Au, GO@Au-PEG and GO@Au-PEG/DOX are all well dispersed in water. After one week of placement, the GO@Au sample had partial precipitation, while the Go@Au-PEG sample had no aggregation precipitation. This indicates that PEG improves the water solubility of GO@Au. In addition, GO@Au-PEG and GO@Au-PEG/DOX was stable in normal saline, medium, fetal bovine serum and PBS solution, and will not accumulate for several weeks.
3.4 Photothermal effect and pH sensitivity
The heating condition of GO@Au under 808 nm laser is shown in Figure 4a. When the water is irradiated by 808 nm laser, the temperature is almost unchanged and there is no obvious thermal effect. The solution temperature of GO and GO@Au increased significantly under the irradiation of 808 nm laser, and the heating efficiency of GO@Au solution was much higher than that of GO solution. This indicates that Au nanoparticles loaded on the GO surface significantly improve the heating efficiency.
The pH sensitivity of PEG in GO@Au-PEG is caused by the break of hydrazone bond in acidic environment, and its pH sensitivity in vitro is shown in Figure 4b. According to the TGA results, the PEG content in GO@Au-PEG nanocomposite was 18.0%. After incubating it in pH 7.4 phosphate buffer solution for 4 h, there was no significant change in PEG content, while after incubating it in pH 5.5 phosphate buffer solution for 4 h, the PEG remaining on GO@Au was only 2.2%. The PEG content of GO@Au-PEG* (without pH sensitivity hydrazone bond) in the control group was 23.8% and 19.6%, respectively, after incubation for 4 h in phosphoric acid buffer solution pH 7.4 and pH 5.5, with no significant change. It is safely to draw the conclusion that PEG in GO@Au-PEG has obvious pH sensitivity.
3.5 GO@Au-PEG/DOX near infrared controlled release
The absorbance of free DOX can be measured by ultraviolet spectrophotometry, and then the content of free DOX can be obtained according to the standard curve. Then the DOX in GO@Au-PEG/DOX can be obtained by difference method, thus the drug loading rate of GO@Au-PEG/DOX can be measured as about 83%.
Photostimulation is an effective stimulus to achieve controlled drug release. In this study, 808 nm laser irradiation of GO@Au was used to generate near-infrared light and release DOX from go@Au-PEG /DO drug delivery system. As shown in Figure 5, under laser irradiation, the DOX release rate from GO@Au-PEG/DOX is very fast. When there is no laser irradiation, the release rate of DOX slows down significantly. At the same time, in the presence of laser irradiation, the DOX release rate of pH 5.5 group was significantly faster than that of pH 7.4 group. This indicates that laser irradiation under acidic conditions is conducive to the rapid release of DOX, which can release 80.1%.
3.6 Cellure uptake
The uptake of MCF-7 cells into the GO@Au-PEG/DOX controlled release system and the near-infrared controlled release of DOX by the system were studied using the red fluorescence DOX as the marker. As shown in Figure 6, GO@Au-PEG/DOX group and GO@Au-PEG*/DOX group after 4 h incubation in the complete medium pH7.4, only very weak burning red light signals, shows that only a tiny amount of DOX into MCF-7 cell. The GO@Au after PEG modification, big space steric of PEG seriously hindered the MCF-7 cell uptake of drug delivery system. Compared with the GO@Au-PEG/DOX (pH7.4) group, the uptake of MCF-7 cells was much faster in the GO@Au-PEG/DOX (pH5.5) group, because PEG was removed due to the pH5.5 rupture of pH-sensitive hydrazone bonds in the weakly acidic environment (pH5.5), and the uptake of GO@Au-PEG/DOX by McF-7 cells was accelerated. GO@Au-PEG*/DOX (without pH-sensitive hydrazone bonds ) did not have this sensitivity, and uptake by MCF-7 cells did not differ significantly between pH 7.4 and pH 5.5. Once inside the cell, DOX is quickly transferred to the nucleus, where it inserts into the DNA and interferes with its synthesis, killing tumor cells. According to Figure 6, the red and yellow light in the GO@Au-PEG/DOX (pH 5.5) group after incubation for 4 h was mainly concentrated in the cytoplasm, indicating that a large amount of DOX was not released from the GO@Au-PEG/DOX drug delivery system. However, when the GO@Au-PEG/DOX (pH 5.5) group entered the cells at 4 h and was irradiated with 808 nm laser for 10 min, strong red light appeared in the nucleus, because the 808 nm laser radiation accelerated the release of a large amount of DOX from the GO@Au-PEG/DOX and the released DOX was transferred to the nucleus quickly. The above results indicated that the weakly acidic environment was conducive to the uptake of GO@Au-PEG/DOX by MCF-7 cells, and the controlled release of DOX by GO@Au-PEG/DOX drug delivery system could be realized by 808 nm laser irradiation group after incubation for 4 h was mainly concentrated in the cytoplasm, indicating that a large amount of DOX was not released from the GO@Au-PEG/DOX drug delivery system. However, when the GO@Au-PEG/DOX (pH5.5) group entered the cells at 4 h and was irradiated with 808 nm laser for 10 min, strong red light appeared in the nucleus, because the 808 nm laser radiation released a large amount of DOX from the Go@Au-PEG/DOX controlled release drug delivery system and was transferred to the nucleus quickly. The above results indicated that the weakly acidic environment was conducive to the uptake of GO@Au-PEG/DOX by MCF-7 cells, and the controlled release of DOX by GO@Au-PEG/DOX drug delivery system could be realized by 808 nm laser irradiation.
3.7 Cytotoxicity of GO@Au-PEG
The effects of GO, GO@Au and Go@Au-PEG at different concentrations on McF-7 cells after 24 h were shown in Figure 7a. As can be seen from the figure, with the increase of GO, go@Au and Go@Au-PEG concentration, the inhibition effect on MCF-7 cells was gradually presented to a certain extent. However, when the concentration of GO, GO@Au and GO@Au-PEG was 100 μg/ml, the inhibition rate of the cells was still less than 15%, indicating that GO, GO@Au and GO@Au-PEG had no obvious toxicity on MCF-7 cells.
Figure 7b shows the effects of GO and G0@AU-PEG at different concentrations on MCF-7 cell growth without and with laser irradiation. In the absence of laser irradiation, the two drugs had little inhibitory effect on cell growth. Under laser irradiation, the growth inhibition of MCF-7 cells in GO group and GO@Au-PEG group increased with the increase of concentration. In addition, at the same concentration, the GO@Au-PEG group had more significant inhibition on cell growth than the GO group, indicating that GO@Au-PEG had stronger photohyperthermia effect under laser irradiation and had a good hyperthermia killing effect.
3.8 Inhibitory effect of GO@Au-PEG/DOX on MCF cell growth
Figure 8 shows the inhibitory effect of DOX and GO@Au-PEG/DOX at different concentrations on the growth of MCF-7 cells under laser and laser-free conditions.As shown in the figure, the growth inhibition of both DOX and GO@Au-PEG/DOX on MCF-7 cells increased with the increase of its concentration. Meanwhile, in the absence of laser irradiation, the growth inhibition effect of DOX at all concentrations on MCF-7 was higher than that of GO@Au-PEG/DOX, which was caused by the slow release of DOX from GO@Au-PEG/DOX. However, under laser irradiation, the growth inhibition of MCF-7 cells by high concentration of GO@Au-PEG/DOX was significantly enhanced, but the effect was not obvious at low concentration, because the heat generated by GO@Au at low concentration was less and could not kill the tumor cells. When DOX concentration was 10 μg/ml, the survival rate of MCF-7 cells in the GO@Au-PEG/DOX group under laser irradiation was only about 10%. This is because both hyperthermia and chemotherapy enhance the therapeutic effect at the same time, the laser promotes the release of DOX, again enhancing the therapeutic effect.
3.9 Tumor growth inhibition in vivo.
In order to investigate the anti-tumor activity of GO@Au-PEG/DOX in vivo, S180 tumor-bearing mice were used as the animal model in this chapter to investigate the tissue distribution characteristics and pharmacodynamic characteristics of the mice.
Several successful tumor-bearing mice were given free water and fasted for 12 hours. Their main organs (heart, liver, spleen, lung, kidney and tumor) and blood were collected separately and weighed, then saline was added to each sample (saline weight: organ weight = 1:1) and homogenized. CO@Au-PEG/DOX (5 mg) was dispersed in 1 ml of water and placed in a dialysis bag. The dialysis bag was then immersed in 10 ml of the homogenates of the different organs and kept in a horizontal laboratory shaker. After 48 h, 500 μl of homogenate was removed, added to a chloroform–methanol mixture (chloroform:methanol = 4:1, 2 ml). After centrifugation at 4000 rpm for 20 min, the chloroform layers were collected and the concentrations of DOX were determined by high-performance liquid chromatography.
As shown in Figure 9, the DOX release in blood, heart, liver, spleen, lung and kidney is very slow, with less than 24% of DOX released after 48 h. The DOX release in Tumor is very fast, with more than 55%of DOX released after 48 h. This indicates that DOX in tumor can be released by the hydrolysis of GO@Au-PEG/DOX on hydrazone bond.
The weight change curve of mice in each group during treatment was shown in Figure 10. Only DOX group mice lost weight, while the weight of other mice did not. The weight loss of mice in DOX group was due to the low selectivity of chemotherapy drug DOX in treatment, which not only killed tumor cells but also caused damage to normal cells in the body, thus causing weight loss. The mice in the GO@Au-PEG/DOX+NIR group lost a little weight (still larger than the initial weight) at the end of treatment, which may be due to the effect of reduced tumor size on weight. At the later stage of treatment, the DOX group and the blank control group showed reduced activity and dull hair color, while the remaining 5 groups showed normal behavior and hair color. The results showed that the GO@Au-PEG prepared in the experiment had low toxicity and could be applied in vivo as a drug transport carrier, which could also improve the selectivity of chemotherapy drug DOX and reduce its toxic and side effects.
Tumor volume changes in each group during administration were shown in Figure 11. During the treatment, tumors in mice of normal saline group, saline + NIR group, GO@Au-PEG group were increased rapidly. At the end of the treatment, tumor volume in the saline group was about three times that at the beginning. No significant changes were observed in saline + NIR group and GO@Au-PEG group compared with the control group. This suggests that NIR irradiation alone could not effectively inhibit tumor growth and GO@Au-PEG alone was not effective in inhibiting tumor growth in the absence of NIR. The tumor volume in the GO@Au-PEG + NIR group at the end of administration was about 2.1 times that at the beginning of administration, indicating that GO@Au-PEG + NIR had a certain hyperthermia effect. Tumor growth was slow in the DOX group, and tumor volume at the end of administration was about 1.9 times that at the beginning of administration, indicating that chemotherapy alone could not effectively treat tumors. The tumor volume in the GO@Au-PEG/DOX group at the end of administration was about 1.1 times that at the beginning of administration, indicating that the GO@Au-PEG/DOX controlled release drug delivery system could deliver DOX to the tumor site with certain tumor targeting. The tumor volume of the GO@Au-PEG/DOX + NIR group at the end of administration was about 52% of that at the beginning of administration, and some of the tumors in mice had been completely eliminated, showing a significant tumor suppressive effect.