3.1 Characterization of GHSO materials
1H-NMR
The 1H-NMR spectroscopy of HA, HSO and GHSO were clearly emerged in Figure 3. The methyl peak for TKL was observed at about δ: 1.5. 1H-NMR spectra revealed absorption peak at about δ: 4.77, which was -OH in ZO. The appearance of the signal peak at δ: 7.1 verified the presence of ZGP, indicating that the new product of GHSO had been synthesized successfully.
FT-IR
FT-IR spectra of GHSO materials were shown in Figure 4. A referred to together dimethyl group, coupled to split into two special shape spectral bands near 1375cm-1, The double-peak in the picture testified the connection of the TKL. B, C referred to skeleton vibration peak of the aromatic ring, D referred to vibration coupling peak of ZO, 1083 cm-1.
3.2 Particle Size, Zeta Potential, Morphology, EE% and DL%
The results of Figure 5 showed that the particle size of HSO@PTX micelles was 143.30±17.00 nm, the PDI was 0.236±0.004, the potential was -23.76±5.12 mV, the encapsulation efficiency (EE%) was 46.47±2.80%, and the drug loading (DL%) was 4.57±0.68%. The particle size of GHSO@PTX micelles was 159.40±14.30 nm, PDI was 0.159±0.06, potential was -24.99±4.73 mV, EE% was 49.61±3.52% and DL% was 4.72±0.39%. The electron microscopy results shown that the two micelles were relatively uniform in size and spherical in shape.
3.3 Drug release investigation
GHSO@PTX at different H2O2 concentrations and pH conditions are shown in Figure 6 below. It can be seen from the figure that each group was slowly released under the conditions and had no sudden release. For the first three groups, the control variable pH remained unchanged at 7.4. As we can see that the H2O2 concentration increased from 0.1 mM to 1 mM, to 10 mM, which was 31%, 38%, and 34% respectively. See that when the H2O2 concentration, the cumulative release increased significantly at 10 mM, for fracture of affinity vector material GHSO under high concentration H2O2, promoting drug release. When the H2O2 concentration was constant and all 10 mM, it showed obvious growth in pH 5.8(76%) compared with pH 7.4(54%), which proved a promoting effect of low pH on nanomicelles. The analysis reason was pH 5.8, ginger acetone bond fracture of GHSO, the conformational change of two affinity carrier materials, micelles dissociation, and promote drug release.
3.4 Cytotoxicity assessment
The cytotoxicity results of nanomicelles to SMMC-7721 cells and CAFs were shown in Figure 7. Figure 7 (E) represented the growth inhibition of blank micelles on both cells at 24 h. It was obvious from the figure that the toxicity of blank micelles is very small, with the survival of 500% at the concentration of 500 μg/ml, demonstrating that the in vivo safety of our designed affinity vector material GHSO was good.
In Figure 7, Figure 7 (A) and (B) represented cytotoxicity results on SMMC-7721 cells by different administration groups at 24 h and 48 h, respectively. Meanwhile, Figure 7 (C) and (D) represented cytotoxicity results on CAFs cells by different administration groups at 24 h and 48 h, respectively. The results showed that with the increase of drug concentration, the survival rate of each drug group decreased to different degrees. At the same drug concentration, the cell survival of nanomicelles group was lower than that of Free PTX, indicating that the nanomicelles group had a stronger lethal effect on tumor cells, probably because HA played the main role in the targeting of tumor cells SMMC-7721. In Figure 7 (C) and (D), the GHSO@PTX group showed the strongest cytotoxicity, possibly due to the fact that it could target CAFs, to enhance the therapeutic effect of tumors.
3.5 Cellular uptake
Figure 8 represented the uptake effect of Free Cur, HSO@Cur, GHSO@Cur on SMMC-7721 cells (A) and CAFs cells (B) at different times. The results in the figure showed that the intake of both cells in each administration group has increased over time. In Figure 8 (A), the nanomicelles group uptake effect was much better than in the Free drug group. In Figure 8 (B), tthe uptake effect of the nanomicelles bunch was superior to the Free medication bunch simultaneously, and the take-up impact of the nanomicelles GHSO@Cur bunch was ideal, additionally in view of its designated impact on CAFs.
Figure 8 (C) and (D) represented the uptake effect at different drug concentrations. It could be seen from the figure that, as the drug concentration increases, the intake effect of each drug administration group has increased to varying degrees. In Figure 8 (C), the uptake of HSO@Cur and GHSO@Cur were better compared with the Free Cur group, proving the superiority of nanomicelles. Another reason was that HA could target the CD44 receptor on the surface of tumor cells, but the two nanomicelles were not significantly different because ZGP was specific for targeting FAPα enzyme on CAFs cell surface and does not promote the uptake of tumor cells. In Figure 8 (D), the nanomicelles group was also better than the free drug group, but the GHSO@Cur group was significantly stronger than the HSO@Cur group because GHSO is specific for targeting CAFs, and thus more uptake superiority, consistent with the initial scenario.
3.6 In vitro penetration of multicellular hybrid tumor spheroids
In this experiment, Cur was selected as a fluorescent substance, loaded in the two affinity vector materials HSO and GHSO, to investigate the in vitro tumor ball penetration effect of two nanomicelles. Figure 9 (A) represented the GHSO@Cur group, Figure 9 (B) represented the HSO@Cur group, and the middle black site represented the site where the nanomicelles failed to reach. It was obvious that in the 3D tumor ball of different particle size, the deep penetration effect of the GHSO@Cur group was significantly stronger than that of the HSO@Cur group. For the analysis reason, it might be that the double-targeted nanomicelles GHSO@Cur group could first target the CAFs, to open the barrier and better achieve the deep penetration of the nanomicelles.
3.7 In vivo fluorescence imaging
The results in Figure 10 shown that the HSO@DiR and GHSO@DiR groups compared with the control Free DiR solution group, DiR can be better delivered to the tumor tissue at about 8 h and accumulate more to the tumor tissue site over time. It was worth noting that the GHSO@DiR group reached the tumor tissue one step earlier than the HSO@DiR group and had a stronger fluorescence intensity at the tumor. At the early stage of administration, the nanomicelles group were distributed at the main organs. Over time, the impact on the main organs had decreased a lot. By 24 h, the double-targeted GHSO@DiR group had the least impact on the main organs, but had the most savings in the tumor site.
The figure on the right represents the results of fluorescence imaging of isolated tissue and organs after administration of 12 h. The results showed that there was no accumulation of the tumor site in the control Free DiR solution group, and both groups of nanomicelles could deliver DiR to the tumor site, demonstrating the superiority of the nanodrug delivery system, and the targeting of HA to tumor cells achieved DiR delivery in the HSO@DiR group. The dual-targeted GHSO@DiR group had the most savings in the tumor site, possibly due to the ZGP targeting of CAFs promoting nanopelels into the tumor tissue. The accumulation of three groups of organs was mainly liver and spleen, the analysis reason might be because the liver as an important metabolic organ of the body, these foreign substances were metabolized by the liver, leading to the accumulation in the liver site; or because preparations are swallowed by macrophages in the body's mesh endothelial cell system, with more macrophages in the liver and spleen, leading in the most accumulation of the liver and spleen parts.
3.8 In vivo pharmacodynamics study
Both Figure10 (A) and 10 (C) could intuitively observe that the HSO@PTX and GHSO@PTX groups are significantly better than the free drug PTX groups, possibly because the nanodrug delivery system could better deliver the drug to the tumor site, improve the effective content of the drug in the tumor site, and play a good therapeutic role, demonstrating the characteristics of pH sensitivity and ROS response. GHSO@PTX group compared with the HSO@PTX group, the treatment effect was better, the analysis might be because GHSO@PTX could specifically target the matrix CAFs, around tumor cells for nanoparticles removal obstacles, promote nanoparticles to better achieve deeper penetration of tumors. The treatment effect of high concentration of GHSO@PTX(+) group was higher than that of GHSO@PTX(-), and there was no naked rat death caused by high concentration during administration, which also affirmed the good compatibility of the carrier material, improved the treatment concentration of the drug and reduced the toxic side effects of the drug. As can be seen from Figure10 (D), the weight of naked rats in the nanopelel group did not change significantly in the early administration, and showed a slight decrease in the later administration, which proved that the nanopelels were less toxic and have certain safety in the body.
3.9 Preliminary histological study
It could be seen from Figure 12 that HSO@PTX and GHSO@PTX had very small damage to the heart, liver, spleen, lung, and kidney of naked rats, combined with the weight change of naked rats, but also more proved the safety of the two affinity carrier materials, reduced the toxicity of drugs, and played a good transport role. It could be seen from the H&E staining results of the tumor tissue of nude tumor rats, there was no damage in the tumor tissue of the saline group, which as a control, observed the free drug group and no excessive damage to the tumor tissue. The analysis might be that the free drug PTX failed to reach the tumor tissue too much and did not play an effective therapeutic effect. Observe the nanosheel group, we found obvious damage to tumor tissue and large death of tumor cells, which proved the inhibitory effect of nanosheel on tumor cells, especially the high concentration GHSO@PTX(+) group, the most loose distribution and obvious core consolidation shrinkage, and proved the best treatment effect of double-targeted nanosheel high concentration GHSO@PTX(+) group.
3.10 Immunohistochemistry
Ki 67 is an important marker of cell proliferative activity. High-level expression of Ki 67 represents a strong cell proliferative activity and can be used as a reliable evaluation indicator after tumor treatment. The results showed that the expression of Ki 67 in the high concentration of GHSO@PTX(+) group was significantly reduced, proving that the tumor cell proliferation activity was significantly inhibited, and that the double-targeted nanogelam GHSO@PTX had a good role in tumor therapy. CAFs cells are α-SMA positive cells, α-SMA staining can brown α-SMA positive cells, and we can judge using the number of brown cells. It can be seen from the figure that, compared with the saline group and the free drug group, the significant reduction of brown cells in the GHSO@PTX group was reduced, indicating that the number of CAFs cells was significantly reduced. The reason analysis may be because GHSO can target the FAP α enzyme on the CAFs surface, and then enter CAFs, to realize the lethal effect on CAFs, thus reducing the promoting effect of tumor microenvironment on the growth of tumor cells. Masson tricolor staining is a classic staining method for collagen cellulose, which dyes collagen cellulose blue. Collagen cellulose is secreted by CAFs cells, deposited in microenvironments, leading to liver fibrosis and promoting the development of tumor cells. From the figure, the blue lines of nanolymical group GHSO@PTX were significantly reduced, indicating decreased collagen secretion, also proving that CAFs is inhibited, while decreased collagen deposition, but also indicating inhibiting the development of liver fibrosis.