2.1. Copolymer synthesis and characterization
VE was conveniently conjugated to HA with an esterification reaction via one synthesis steps in the presence of EDC and DMAP, as demonstrated in Fig. 1A. The synthesis of HA-VE conjugate was confirmed by 1H NMR analysis. As shown in Fig. 1B, the characteristic peaks for methylene groups at the sugar unit and N-acetyl group of HA were identified at 3.10 ~ 4.00 ppm and about 1.80 ppm [20]. Intense peaks appeared at 0.8 ppm and 1.00 ~ 2.00 ppm represented the methyl group and methylene on fatty acid chain of VE [21]. These characteristic peaks could be observed in the 1H NMR of HA-VE conjugate, indicating that it was successfully synthesized.
PBAEss were synthesized with the yield of 72.3%, mediated by Michael addition reaction as shown in Fig. 1C. The molecular weight of PBAEss copolymer was determined by gel permeation chromatography (GPC) analysis (Fig. S1). The Mw and Mn of PBAEss was 3.51×104 and 1.75×104, respectively (Mw/Mn = 2.00). The chemical structures of these intermediates, including 4-amino-1-butanol, HDD and BACy, and the synthesized PBAEss copolymer were characterized by 1H NMR spectra (Fig. 1D). As shown, the characteristic peaks at 4.2 ppm and 5.5 ~ 6.5 ppm in HDD spectrum attributed to –OCH2- and vinyl protons. In BACy spectrum, the peaks at around 3.40 ppm and 5.5 ~ 6.5 ppm were assigned to the proton shifts of –SCH2– and the characteristic protons of vinyl protons, respectively. However, after polymerization between the vinyl of HDD and BACy, and amine group of 4-amino-1-butanol, the chemical shift derived from vinyl protons were disappeared in PBAEss spectrum. Meanwhile, the characteristic methylene proton peaks of NH2CH2-, -CH2-CH2-, and -CH2OH in 4-amino-1-butanol appeared respectively at 2.46 ppm, 1.2 ~ 1.5 ppm, and 3.4 ppm also could be found in the 1H NMR spectrum of PBAEss. Altogether, the above results indicated PBAEss was successfully synthesized.
2.2. Preparation and characterization of TPL/NPs
PBAEss analogues have been previously demonstrated to form NPs for efficiently encapsulating genes or anticancer drugs by self-assembly based on its amphiphilic property. Herein, we encapsulated TPL into PBAEss nanocore using the nanoprecipitation method. Meanwhile, the hydrophilic HA-VE shell was coated on PBAEss nanocore by electrical charge interaction. To obtain the NPs with high drug loading efficiency and suitable size distribution, different compositions of HA-VE and PBAEss copolymer were utilized (Table S1). Conclusively, HA-VE, PBAEss, TPL with the amount of 10 mg, 10 mg, and 2 mg were feed. The collected TPL-loaded in HA-VE/PBAEss NPs (TPL/NPs) exhibited an average diameter of approximately 191 nm with a relatively narrow distribution (Table 1). The drug loading efficiency (DLE) and drug entrapment efficiency (DEE) of TPL loading in NPs were determined as 8.63 ± 0.72% and 94.93 ± 2.1%, respectively.
The size distribution of TPL/NPs by dynamic light scattering (DLS) and morphology by Transmission Electron Microscopy (TEM) observation were shown in Fig. 2A. As shown, TPL/NPs had a uniform size distribution, regularly spherical shape and a compact structure. However, in response to either reductive (GSH 10 mM) or acid condition (pH 5.8) to mimic the redox and acid tumor microenvironment, the size distribution of TPL/NPs began to increase with multiple peaks. The uniform orbicular morphology of TPL/NPs changed into dispersion and fragment. The size changes of TPL/NPs in response to different conditions were shown in Fig. S2. These above results demonstrate that the TPL/NPs with the high drug loading and narrow particle size possessed the pH- and redox- dual responsive properties, which was suitable for the controlled release profiles in tumor microenvironment. Moreover, as depicted in Fig. S3, TPL/NPs could remain a stable particle size below 200 nm either in pH 7.4 PBS or in 10% serum supplemented DMEM at 4°C until 96 h place. After the continuous storage for 14 d, TPL/NPs exhibited the stable size distribution, with the RSD values of average size of 3.3% (Fig. S4). These results indicated the good stability of TPL/NPs, attributed to the uniform size distribution and anionic-based electrostatic repulsion.
To evaluate the intermolecular interaction between TPL and HA-VE/PBAEss nano-vehicle, fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) were employed. In Fig. 2B, the strong characteristic peak at 3455 cm− 1 and 1770 cm− 1 were shown in the FT-IR spectrum of TPL (a), derived from the hydroxyl group and carbonyl group in lactonic ring, respectively. Mediated by none of interference on these signals in FT-IR spectrum of blank NPs (b), the disappearance of these peaks in TPL/NPs (c) on the same chemical shift suggested that TPL was totally encapsulated in polymeric NPs, instead of physical mixture. Additionally, in the XRD spectrum (Fig. 2C), due to the crystal structure of TPL, the characteristic crystallization peaks mainly arranged at the range of 10°~40°. After loading in NPs, TPL/NPs transformed into amorphous form, indicating that there were no crystalline peaks in TPL/NPs.
To ensure the biosafety of TPL/NPs for intravenous injection, hemolysis analysis experiment was also conducted. We examined the effect of TPL/NPs on the hemolytic profile of RBC. PBS and water were used as the negative and positive control. As expected, the TPL/NPs with 1 ~ 50 µg/ml would not drive erythrocytes to release hemoglobin with a hemolysis rate of ≤ 5% (Fig. 2D). It indicated the safety of TPL/NPs and its compatibility for I.V. administration.
Table 1
Characterization of TPL/NPs
Samples | Particle size (nm) | PDIa | Zeta potential (mV) | DLE (%) | DEE (%) |
Blank NPs | 184.53 ± 7.52 | 0.195 ± 0.052 | -6.33 ± 0.32 | - | - |
TPL/NPs | 191.31 ± 7.08 | 0.176 ± 0.040 | -6.94 ± 0.58 | 8.63 ± 0.72 | 94.93 ± 2.1 |
a Polydispersity index |
2.3. In vitro drug release of TPL from TPL/NPs
In view of the pH/redox sensitive properties demonstrated by size changes above, the in vitro TPL release profiles from TPL/NPs under different mediums were measured using dialysis method, including the physiological condition (pH 7.4), only redox condition (pH 7.4, 10 mM GSH), only acid condition (pH 5.8), simulative tumor intracellular condition (pH 5.8, 10 mM GSH). Free TPL could be released rapidly as control, in which the cumulative release of TPL could reach about 90% of TPL during 8 h (Fig. 2E). At pH 7.4, TPL in NPs was released gently, only 45% of TPL was released from NPs druing 24 h, indicated the TPL molecules could be protected well in the core of HA-VE/PBAEss NPs. In comparison, TPL/NPs in response to either 10 mM GSH or pH 5.8 buffer exhibited much faster drug release, compared to that in pH 7.4 buffer. In response to 10 mM GSH, the PBAEss copolymer was expected to fracture due to the cleavage of disulfide bond. And when the pH value was decreased to 5.8, the drug release rate was sharply accelerated due to the protonation of tertiary amine residues in PBAEss segment. Therefore, the cumulative release of TPL for 24 h was 71.65% and 76.31%, respectively, indicated the intense initial burst release. At pH 5.8 with 10 mM GSH, the cleavage of disulfide bond and protonation of tertiary amine were combined, so that the drug release exhibited the fastest profile. The cumulative release of TPL reached to 85.32% during 24 h. Taken together, TPL-loaded in HA-VE/PBAEss NPs can efficiently exhibit a controlled release in physiological condition and a selectively rapid drug release in tumor microenvironment.
2.4. Cytotoxicity against breast cancer cells of TPL/NPs
After 48 h incubation, we investigated in vitro cytotoxicity of TPL/NPs in different media, i.e., pH 7.4, pH 7.4 + 10 mM GSH, pH 5.8, pH 5.8 + 10 mM GSH, respectively, against MDA-MB-231 and MCF-7 cells by MTT assay. The pH 5.8 value and 10 mM GSH of culture medium were characterized as the early endosome and cytoplasm of cancer cells. All these TPL formulas suppressed proliferation of MDA-MB-231 and MCF-7 cells in a dose-dependent manner at the range of 0 ~ 160 nM, whereas Blank NPs without TPL loading will not induce the cell viability reduction (Fig. 3, Fig. S5). Under the media of pH 7.4, TPL/NPs exhibited slightly higher cellular proliferation inhibition than free TPL, the IC50 values of TPL/NPs in both MDA-MB-231 and MCF-7 cells were close to that of free TPL. However, TPL/NPs showed much higher cytotoxicity at all concentrations of TPL exposed to these other culture media. Specifically, the average IC50 value of TPL/NPs in MCF-7 and MDA-MB-231 cells under pH 7.4 medium was 58.67 nM and 72.28 nM, while it remarkably reduced into 20.76 nM and 41.77 nM under the media of pH 7.4 + 10 mM GSH ,reduced into 18.11 nM and 34.14 nM under the media of pH 5.8. Nevertheless, the IC50 values of TPL/NPs under the medium of pH 5.8 + 10 mM GSH were minimum, which sharply decreased into 13.77 nM and 26.39 nM in MCF-7 and MDA-MB-231 cells, respectively (Fig. 3C). These results are well consistent with the in vitro drug release profile, indicated that the pH-/redox- sensitive NPs facilitate the drug release and efficiently kill the tumor cells.
2.5. TPL/NPs enhanced the cell apoptosis and cell cycle arrest
Apoptosis is the main mechanism accounting for the anticancer of TPL [22]. Herein, we further evaluated whether TPL/NPs would enhance the apoptosis in both MDA-MB-231 and MCF-7 cells. To thoroughly mimic the acid and reductive microenvironment in tumor, we performed the apoptosis induction of TPL/NPs under pH 7.4 without GSH or pH 5.8 with 10 mM GSH. Apoptosis was analyzed by annexin V-FITC / PI staining. As shown in Fig. 4, with the equivalent TPL amount of 20 nM for 24 h incubation of MDA-MB-231 cells, TPL/NPs at each condition exhibited higher apoptosis rate than free TPL. Especially, TPL/NPs in culture medium of pH 5.8 with 10 mM GSH induced the highest apoptosis rate, compared to other counterparts. The apoptosis rate of TPL/NPs at pH 5.8 with 10 mM GSH was 2 and 1.6 -fold of that of free TPL and TPL/NPs at pH 7.4, respectively. Moreover, the results in MCF-7 cells were also in accordance with that in MDA-MB-231 cells.
Cell cycle phase distribution was analyzed by flow cytometry (FCM) with PI staining, which could measure the cellular DNA content. As shown in Fig. 5, after 24 h incubation in MDA-MB-231 cells with 10 nM TPL, a high proportion of cells in Sub-G0/G1 was observed, which indicated the occurrence of cell apoptosis. It was in accordance with the previous report about the SubG1 phase arrest of TPL [23]. Meanwhile, more either MCF-7 or MDA-MB-231 cells were significantly arrested in G0/G1 phase when treated with TPL/NPs, compared to that treated with free TPL. As expected, the TPL/NPs in the medium of pH 5.8 + 10 mM GSH also induced a significantly higher G0/G1 population than that in the medium of pH 7.4.
In summary, TPL/NPs exhibited the stronger pro-apoptosis and G0/G1 cell cycle arrest capacity in both MDA-MB-231 and MCF-7 cells compared to free TPL. In view of the stimuli-responsive drug release of TPL/NPs, both the cell apoptosis and cell cycle arrest of TPL/NPs exhibited the significant enhancement in response to pH-/redox- culture medium, suggesting that TPL/NPs benefited to play its anticancer effect in tumor microenvironment.
2.6. Cellular uptake study
To determine whether the TPL/NPs specifically enhanced cellular uptake of breast cancer cells, we measured the uptake of C6/NPs in MDA-MB-231 and MCF-7 cells after being treated for 1, 2, and 4 h by FCM. After incubation with free C6 and C6/NPs, the accumulation of free C6 and C6/NPs in MDA-MB-231 and MCF-7 cells was time-dependent (Fig. 6A and 6B). Besides, after co-incubation with cells, cellular uptake of C6/NPs was significantly greater than that of free C6 at different time intervals, indicating enhanced cellular uptake of nanosize agents. The enhanced cellular uptake of C6/NPs was mediated by active transport in an energy dependent manner (Fig. S6A), rather than the passive diffusion of free hydrophobic C6. Additionally, drugs loading in NPs could avoid being easily pumped out from cells.
Besides, we also observed the cellular uptake of C6/NPs both in MDA-MB-231 and MCF-7 cells by confocal laser scanning microscopy (CLSM) (Fig. 6C and 6D). After treatment for 4 h, higher cellular internalization of C6 was found in cells treated with C6/NPs than in cells treated with free C6. These data were consistent with FCM results, indicating that C6/NPs were effectively taken up.
2.7. Competitive uptake study
To investigate the CD44-targeted delivery of the TPL/NPs, CD44-positve MDA-MB-231 breast cancer cell line and CD44-negative MCF-7 epithelial cell line were chosen (Fig. S7)[24]. Figure 6C and 6D taken by CLSM showed the intracellular uptake of C6/NPs in CD44-positive MDA-MB-231 cells and CD44-negative MCF-7 cells. The cell nuclei was stained with Hoechst 33342, which exhibited strong blue fluorescence. Compared to free C6 group, we observed a more intense fluorescence signal in MDA-MB-231 and MCF-7 cells when incubated with C6/NPs. However, when C6/NPs group was pretreated with free HA for 1 h (C6/NPs + HA), the fluorescence expression was significantly decreased in MDA-MB-231 cells, which indicated that free HA blocked the CD44 receptor on the surface of MDA-MB-231 cells and inhibited internalization of C6/NPs. On the other hand, when C6/NPs group was pretreated with 5 mg/ml of free HA for 1 h (C6/NPs + HA), the fluorescence expression was slight reduced in MCF-7 cells. The results indicated that HA-modified NPs could specifically bind to the CD44 receptor on the surface of tumor cells and promote cellular uptake by active targeting.
Furthermore, the competitive study was studied in CD44 high-expressed MDA-MB-231 cells by FCM. The cells were pretreated with free HA followed by incubated with C6/NPs. The result is shown in Fig. S6B where the cellular accumulation of C6/NPs was reduced to 48.58 ± 3.7%. It demonstrated that the cellular uptake of C6/NPs was contributed to HA polymer specific binding to CD44 receptors. The free HA competed with HA-conjugated NPs for CD44 receptors and inhibited cellular uptake of HA-conjugated NPs in cells.
2.8. Endocytosis pathway
We utilized different endocytic inhibitors to identify the possible endocytotic pathways involved in the uptake of TPL/NPs, including chlorpromazine (clathrin-mediated), indomethacin (caveolae-mediated), methyl-β-cyclodextrin (cholesterol-dependent endocytosis) and colchicine (macropinocytosis inhibitor) [25]. As shown in Fig. 6E, the internalization of C6/NPs was significantly decreased by the pretreatment of indomethacin or methyl-β-cyclodextrin, indicating the process of internalization of C6/NPs was involved in caveolae-mediated and cholesterol-dependent endocytosis. In contrast, the pretreatment of neither chlorpromazine nor colchicine could exert little effect on cellular uptake of C6/NPs, excluding the involvement of clathrin-mediated endocytosis and macropinocytosis.
2.9. TPL/NPs inhibited MDA-MB-231 cell migration and invasion
The MDA-MB-231 cell line as the triple negative breast cancer cell possesses high metastatic character [26]. We conducted wound-healing assay to evaluate whether the TPL/NPs treatment could suppress the migration of MDA-MB-231 cells. The results indicated that the wound gaps in the TPL/NPs-treated groups were significantly wider than either those of the untreated groups or those of free TPL treatment group, indicated that TPL/NPs could more effectively inhibit the migration of MDA-MB-231 cells compared to free TPL (Fig. 7A and 7B). Particularly, in the medium of pH 5.8 with 10 mM GSH, the migration inhibition of TPL/NPs was remarkably higher than other counterpart.
Furthermore, the anti-metastasis effect of TPL/NPs was evaluated by transwell experiments. Consistent with the results of the wound healing assay, TPL/NPs effectively suppressed cell migration. Only a few MDA-MB-231 cells migrated from the upper chamber to the lower chamber of the transwell following the treatment with TPL/NPs in pH 5.8 + 10 mM GSH, indicated that TPL/NPs significantly inhibited the longitudinal motility of MDA-MB-231 cells (Fig. 7C and 7D).
To study the effect of TPL/NPs on cell invasion, MDA-MB-231 cells were treated with either free TPL or TPL/NPs, then allowing cells to invade in Matrigel-coated Transwells for 24 h. The number of cells that invaded was decreased by various TPL formulas (Fig. 7C and 7E). Compared to the untreated group, free TPL, TPL/NPs in pH 7.4, and TPL/NPs in pH 5.8 + 10 mM GSH inhibited invasion by ~ 45%, 52%, and 67%, respectively. The results clearly demonstrated that TPL/NPs could strongly suppress MDA-MB-231 cell migration and invasion, with the pH-/redox- dual sensitive combination effects. These above results implied the anti-metastasis potential of TPL/NPs.
2.10. In vivo biodistribution of HA-VE/PBAEss NPs
The tumor targeting capacity of HA-VE/PBAEss NPs was further investigated in 4T1 tumor bearing nude mice. To label the HA-VE/PBAEss NPs with fluorescence, DiR was firstly encapsulated into NPs with the similar approach as TPL as shown in Fig. 8A. When the tumor volume reached about 1000 mm3, the mice were administrated intravenously with free DiR and DiR/NPs. The mice were imaged using the IVIS® Spectrum scanner during 72 h post-injection (Fig. 8B). At 72 h, the mice were sacrificed, and the main organs as well as tumor were harvested and visualized ex vivo (Fig. 8C). As shown, after administration of free DiR by i.v., the NIR fluorescence in vivo attenuated sharply, and few of fluorescence could be found in tumor tissue, indicated that the rapid body clearance and poor tumor accumulation capacity. However, much more fluorescence with a prolonged period can be found in mice treated with DiR/NPs. Particularly, the significant tumor accumulation of DiR/NPs could be observed. Even after 72 h post-injection, strong fluorescence still could be found in tumor tissue. After injection of DiR/NPs for 72 h, the strong signal can be observed in tumor, instead of the unconspicuous fluorescence of tumor in free DiR group. This indicated that the tumor accumulation of vesicles composed by HA-VE/PBAEss copolymers, which would attribute to the prolonged circulation, EPR effect and tumor targeting mediated by HA and CD44 interaction. Taken together, the results indicated that the in vivo long-circulation and tumor accumulation of TPL can be efficiently enhanced by the capsulation of HA-VE/PBAEss NPs.
2.11. Inhibition of primary tumor growth and metastasis in vivo
Mediated by the combination of tumor-homing properties and dual stimuli-triggered tumor release profiles of TPL/NPs, we assumed that it could be much more efficient in chemotherapy. Therefore, we established a 4T1 subcutaneous tumor bearing mouse model to further evaluate the in vivo efficacy in suppressing primary tumor growth and distant metastasis. After treated with different administrations for 20 days, the tumor growth curves (Fig. 9A) and body weight (Fig. 9B) were presented, which were measured every two days. As shown, the blank HA-VE/PBAEss NPs shown no distraction on tumor growth as well as body weight, similar to saline control group. However, mice treated with TPL formulations exhibited better antitumor activity, compared to saline group. The tumor volume inhibition rates for TPL, TPL/NPs (L) and TPL/NPs (H) were 54.86%, 70.10%, and 84.76%, respectively. Particularly, the TPL/NPs at either 0.2 mg/kg or 0.4 mg/kg exhibited higher tumor growth inhibition efficacy than free TPL at 0.4 mg/kg. The TPL/NPs (H) almost inhibited the bearing tumor tissue growth completely. Meanwhile, after a successive administration of free TPL for 14 days, mice exhibited the declined body weight, indicated the potential side-effects. Nevertheless, after loading in NPs, TPL exhibited the higher biosafety. The differences among groups on inhibition effects to primary tumor can be directly observed in Fig. 9C. At the experimental end point, tumor weight in each group had the same trend with the result of tumor volume (Fig. 9D). These results demonstrated that TPL/NPs (H) with the TPL of 0.4 mg/kg possessed the best curative effect on primary tumor and non-side effects.
The 4T1 subcutaneous tumor has been demonstrated to be prone to induce pulmonary metastasis. In view of the cell migration and invasion inhibition effects in vitro, the anti-metastasis ability of TPL/NPs in vivo was also evaluated. After perfusing the lungs with ink gelatin solution, the pulmonary metastatic nodules could be visible. A large number of metastatic nodules were observed in the lungs of the saline and blank NPs groups (Fig. 9E). Compared with the saline group, the number of pulmonary metastatic nodules in mice treated with TPL, TPL/NPs (L) and TPL/NPs (H) decreased by 55.38%, 67.70%, and 89.99%, respectively (Fig. 9F). The number of pulmonary metastatic nodules in TPL/NPs (H) group were remarkably less, compared to other counterparts. These results corroborated that TPL/NPs could efficiently suppress primary tumor growth and pulmonary metastasis.
2.12. Potential anti-tumor and anti-metastasis mechanisms
To further investigate the mechanisms of proliferation and metastasis inhibition of TPL/NPs in vivo, the tumor tissues were implemented by hematoxylin and eosin (H&E) and immunohistochemical staining (Fig. 10A). The H&E images indicated that the TPL/NPs induced the limited proliferation and severe apoptotic damages, even necrocytosis, in the tumor compared to either free TPL or blank NPs. This result was also confirmed by the immunohistochemical staining of Bcl-2 and Ki-67 antibody. In TPL/NPs (H) group, the number of brownish yellow points represented the positive cells was much fewer than other counterparts. In our previous study, we have demonstrated that TPL could significantly suppress the angiogenesis, benefiting to block the tumor growth. Herein, the expression of CD31 was significantly downregulated in TPL/NPs (H) group compared with other groups, indicated the higher anti-angiogenesis activity of TPL/NPs. Moreover, the expression of MMP-9 in the tumor tissue possesses a close relationship with tumor invasion and metastasis. As shown, the expression of MMP-9 was uniform over the whole slice of saline group, while the lowest expression of MMP-9 was observed along with the treatment of TPL/NPs (H). E-cad as an important EMT-relative protein plays a pivotal role in many epithelial malignant tumors invasion and metastasis. Invasion of surrounding tissues and metastasis have been proposed to initiate following loss of E-cadherin (E-cad) [27]. As expected, low expression of E-cad could be found in saline group, while the remarkably increased E-cad expression appeared in the TPL/NPs (H) group. The downregulated MMP-9 and upregulated E-cad indicated that after treatment with TPL/NPs, the metastasis process was significantly restrained (Fig. 10B). The similar results were also observed in histological staining images of lung tissues by H&E and immunohistochemical staining (Fig. S8). Because of the generation of pulmonary metastatic nodules, the cell proliferation, blood vessel and cell migration in lung tissue of saline group exhibited extremely active. However, the treatment of TPL/NPs could effectively callback the abnormal upregulation on the expression of these representative proteins including Bcl-2, Ki-67, CD31, MMP-9, and the decreased E-cad amount in lung tissue. These results were consistent with the conclusion obtained from tumor tissue of immunohistochemical analysis.
Furthermore, to validate the regulation effect on tumor growth and metastasis of TPL/NPs, western blotting analysis was also performed to assess the expression of some apoptosis and metastasis signaling proteins, including p53, caspase 3, MMP-2, and vimentin. As shown in Fig. 10C and 10D, TPL/NPs significantly upregulated the protein levels of p53 and caspase 3, compared to either control group or free TPL group. Therefore, combined with the regulation of Bcl-2, caspase 3 and p53, the data demonstrated that TPL/NPs significantly induced tumor apoptosis mediated by mitochondrial apoptotic pathway. In addition, the representative proteins in tumor EMT process including MMP-2 and vimentin were evaluated by western blot. As expected, the levels of MMP-2 and vimentin were remarkably decreased in TPL/NPs groups (Fig. 10D). In view of the regulation of E-cad, MMP-9, MMP-2 and vimentin, the results shown that TPL/NPs effectively suppressed tumor metastatic effects in vivo.
Although the outstanding antitumor growth and anti-metastasis effect of TPL/NPs have been observed, whether TPL/NPs could result in the side-effect was still unclear, in view of the acknowledged severe side-effects of TPL. Therefore, we evaluated the histological images of the main organs by H&E staining and some blood biochemical indexes. Compared to saline group, free TPL-treated mice showed histopathological changes in liver tissue, characterized by fat vacuoles, inflammatory cell infiltration and necrosis (Fig. 11A). It indicated the liver injury caused by free TPL administration. However, the severity of histopathological lesions in TPL/NPs groups were less than those in the free TPL group. Furthermore, significant increases in alanine aminotransferase (ALT) and aspirate aminotransferase (AST) levels in blood were observed in mice serum treated with 0.4 mg/kg of the free TPL compared to saline group (Fig. 11B). In contrast, the administration of TPL/NPs with each dosage don’t lead to significant changes in the levels of those indexes. However, there were no obvious differences in the levels of urea nitrogen (BUN) and creatinine (Cr) among all groups.