Preparation and characterization of honokiol-loaded MSNPs (HNK-M)
Hydrophobic drug honokiol was encapsulated in hydrophilic silica nanoparticles. The HNK-M compound can improve the efficacy of honokiol and cellular uptake compared to the free-honokiol. MSNPs have hydrophilic surfaces and are easier to be absorbed by cells. Besides, MSNPs are acknowledged to be one of the most promising drug-carrier systems because it can be readily removed from the body and is nontoxic. Small molecule drugs (e.g., honokiol in this study) were usually loaded into MSNPs by three generic strategies, i.e., the molecules of honokiol are embedded into dense interior of MSNPs, encapsulated in the mesoporous structure of MSNPs, or physically adsorbed on the surface of MSNPs . We prepared HNK-M with a conventional method in our laboratory and evaluated its morphology and also the toxicity to VSMCs. Figure 2a showed a typical transmission electron microscopy (TEM) image of HNK-M. They were spherical in shape and superimposed on top of each other. The density inside HNK-M is not uniform as a result of the presence of mesoporous interspace. The WST-1 assay was used for quantitative testing of the viability of VSMCs treated with free-honokiol, MSNPs and HNK-M. WST-1 is a compound similar to MTT and can be reduced to orange formazan by dehydrogenase in the mitochondria. It was shown that the amount of formazan dye is directly correlated to the number of living cells . As shown in Figure 2b, MSNPs is almost nontoxic to VSMCs. The optimal concentration of honokiol for effective therapy on VSMCs was believed to be around 100μM. Figure 2b showed that there was no difference in toxicity between free-honokiol and HNK-M at concentrations ranging from 80μM to 120μM. The IC50 values for free-honokiol and HNK-M were 136.8 and 152.7μM, respectively. These results provided that MSNPs effectively delivers the honokiol to VSMCs by virtue of its preferable biocompatibility and reduce the toxicity of the honokiol to cells. Thus, to avoid potential interference with cell viability, the non-cytotoxic concentrations of honokiol (≤136.8μM) were used in the subsequent experiments.
HNK-M inhibits VSMCs growth in vitro
PCNA is an accessory protein of DNA polymerase delta and is required in the synthesis of DNA during the cell cycle . Thus, PCNA can be used as an indicator to evaluate cell proliferation. In order to evaluate the inhibition effect of HNK on proliferation of VSMCs, flow cytometry was used to determine the expression of PCNA in VSMCs treated with free-honokiol and HNK-M respectively. As shown in Figure 3a-b, the downregulation of PCNA expression in VSMCs treated with MSNPs was similar to that in blank control group, indicating that MSNPs has no inhibitory effects on proliferation of VSMCs. Over the same concentration range, the downregulation rates of PCNA in the free-honokiol group were 7.4% and 14.2% respectively at concentration of 80 and 120μM. However, the results of the HNK-M group were 17.1% and 24.5%, which were both larger than that with corresponding concentrations in free-honokiol group. Taken together, these results suggest that the application of MSNPs significantly increased the ability of honokiol to inhibit the proliferation of VSMCs. In addition, the dependence of PCNA inhibition on concentration of honokiol was also observed both in free-honokiol and HNK-M group.
HNK-M inhibits VSMCs migration
As described above, migration of VSMCs to the intima is one of the mechanisms leading to restenosis after vascular injury. To test whether HNK-M can inhibit the migration of VSMCs, we performed a transwell assay on cultured VSMCs treated with HNK, MSNPs and HNK-M. Transwell assay was carried out by Boyden chamber. It consists of two chambers separated by a microporous membrane as a physical barrier so that cells can only overcome by active migration. As shown in Figure 3c-d, on the average, there were 78.5 cells crossing the membrane in control group. While in both free-honokiol and HNK-M group, fewer migrating cells were observed. When compared with that in free-honokiol group, HNK-M significantly reduced the number of migrating cells both at 80 and 120μM concentration. We also found that HNK-M inhibited VSMCs migration in a concentration-dependent manner. These results confirmed that HNK-M can show an efficient inhibition on VSMCs migration.
HNK-M down-regulates proteins involved in TGF-β signaling pathway
The regulation of TGF-β on VSMCs proliferation is related to a variety of downstream pathways, e.g., the activation of the samd family signaling pathway . To evaluate the potential inhibitory effects on TGF-β signaling pathway, western blots were performed on p-smad3 of VSMCs treated with free-honokiol, MSNPs and HNK-M. As shown in Figure 4a-b, overexpression of p-smad3 on VSMCs was observed by irritation with TGF-β (5ng/ml) for 1h. The phosphorylation state of smad3 can be inhibited by free-honokiol and HNK-M, and the inhibitory effects of HNK-M is more obvious than that of free-HNK. Beyond that, we further evaluated the interaction of smad family and ERK pathway in VSMCs and explored whether HNK-M downregulated the expression of PCNA induced by p-ERK. VSMCs were cultured with SIS3 (SIS3 is a cell-permeable and selective inhibitor of smad3) or PD98059 (PD98059 is a potent, selective and cell-permeable MEK1 and MEK2 inhibitor) and pretreated with free-honokiol, MSNPs and HNK-M for 24h, respectively. The expression of p-ERK and PCNA were measured with western blot assay. In Figure 4c-f, we confirmed that the expression of activated ERK1/2 decreased when inhibiting the phosphorylation process of smad3, and HNK-M can significantly down-regulate the expression of p-ERK1/2 and PCNA compared to the free-honokiol group.
HNK-M reduces PCNA expression in vitro
PCNA, a protein synthesized in early G1 and S phases of the cell cycle, maintains at a high level in proliferation cells . In most cases, signaling pathways promote cell proliferation by inducing PCNA expression. In this study, using RT-PCR, we found that HNK-M reduced the expression of PCNA mRNA in VSMCs when compared with other groups (Figure 5a). To further evaluate the expression of PCNA protein, IF technology was utilized to detect the fluorescence intensity of PCNA in each group. As shown in Figure 5b-c, VSMCs in control group expressed highest levels of PCNA, while free-honokiol and HNK-M groups showed a remarkably lower fluorescence intensity of PCNA. At the same concentration, the fluorescence intensity of PCNA in HNK-M group was lower than that in free-honokiol group, which indicates the significance of MSNPs carrier for HNK delivery.
HNK-M inhibits intimal hyperplasia after vascular injury
In order to determine the point-in-time of draw materials after drug administration to animals, degree of intimal was measured after common carotid artery injury at day 3, 7, 14 and 28. As shown in Figure 6c, there was no visible hyperplasia of intima after 3 and 7 days of vascular injury, so it was impractical to evaluate the changes of intima during this period. The new neointima was developed enough at 14 days after injury, and the neointima formation process kept at a stable state compared with 28 days, this is consistent with previous research . Hence, 14 days after carotid injury operation was set as the predetermined timepoint in the following study to draw materials from the rats.
Intimal hyperplasia is the primary cause of restenosis after vascular injury . The potential of HNK-M in prevention and treatment of restenosis after vascular injury can be preliminarily evaluated by investigating the intimal thickness 14 days after balloon injury. We applied a HE staining to determine the intimal situation in this rat balloon injury model. As shown in Figure 7a, in the control group, the neointimal hyperplasia caused by balloon injury induced significant stenosis of the artery, which almost blocked the lumen. Compared with the control group, HNK-M administration can markedly inhibit the intimal hyperplasia, which was then quantified by increased intima/media ratio and intima area. In figure 7b-c, at 14 days, there was a 72.5% decrease in the intima area of HNK-M group compared with that of free-honokiol group (0.2651±0.024 vs 0.0728±0.009, P=0.0019). Meanwhile, the intima/media ratio decreased by 64.5% (0.1520±0.005 vs 0.0539±0.007, P=0.0004). However, no differences were observed in intimal area between control group and MSNPs group. The results indicated that HNK-M administration had a higher inhibition on the intima hyperplasia in the injured vascular compared with free-honokiol.
HNK-M inhibits collagen deposition
Activation, aggregation and adhesion of platelets were induced at the site of injury after endothelial injury. The activated platelets secrete growth factors and cytokines which then induce VSMCs migration, proliferation and ECM synthesis. This is followed by cellular proliferation and migration and the expression of ECM proteins leading to the formation of neointima and restenosis after stent implantation or angioplasty [15, 36]. Collagen is the main component of ECM and we have previously shown that free-honokiol inhibits collagen deposition in injured carotid artery. The position rate of intimal collagen was measured by masson staining after 14 days of damaged vessels with drug administration. Figure 7d-e revealed that collagen expression was upregulated in the neointima of drug treatment group. However, the deposition of collagen in vascular intima was less in the HNK-M group than the free-honokiol group (P＜0.05), which indicated a higher inhibition of HNK-M on collagen deposition.
HNK-M inhibits VSMCs proliferation in vivo
In normal vascular tissue, VSMCs have a contractile phenotype: they proliferate slowly, are functionally contractile and express a range of contractile proteins, including α-SM-actin (α-SMA)  . We investigated whether HNK-M could suppress expression of PCNA and VSMCs proliferation in vivo using a rat carotid artery balloon-injured model. We performed a immunofluorescence staining for α‑SMA and PCNA, and used α‑SMA to localize VSMCs in the intima. As presented in Figure 8a-b, after 14 days of injury, the immunofluorescence of the blood vessels showed that most cells in the neointima expressed α‑SMA (green). PCNA expression in VSMCs of HNK-M treatment group was significantly down-regulated compared with the control group (P=0.0067), and yet, there was no difference between the free-honokiol group and the control group (P=0.0898). These data suggest that HNK-M significantly reduced the proliferation ability of VSMCs in the intima compared to the free-honokiol.
HNK-M inhibits the conduction of the TGF-β signaling pathway in vivo
New data from in vivo and in vitro studies have shown that TGF-β plays an important role in the regulation of a key pathological response to restenosis: intimal thickening and arterial remodeling . We have demonstrated in vitro that TGF-β can stimulate the phosphorylation of smad2/3 in VSMCs, and overexpression of p-smad2/3 can also activate ERK1/2. Therefore, in in vivo experiments, we also used western blot to evaluate the expressions of TGF-β, p-smad2/3 and p-ERK1/2 in common carotid artery tissues at predetermined time after ballon-injuryed carotid artery in rats. As shown in Figure 8c-f, at 3days, 7days and 14days after balloon injury, free-honokiol and HNK-M had no effect on the expression of TGF-β, but inhibited the expression of p-smad2/3 and p-ERK1/2, and the inhibitory effect of HNK-M was slightly stronger than that of the free-honokiol. This result demonstrated that p-smad2/3 and p-ERK1/2 expression was increased from the 3rd day after endovascular injury, and could continue until the 14th day. The in vivo and in vitro results are consistent, further demonstrating that the free-honokiol inhibits VSMCs proliferation by inhibiting the TGF-βpathway, and that the effectiveness of the free-honokiol can be enhanced by encapsulated with MSNPs.