ZnO NPs-mediated uptake enhancement and efflux attenuation
In our previous study, we successfully loaded DOX onto the ZnO NPs and studied the influence of the ZnO NPs on DOX’s anticancer activity [22]. In this study, the similar method was used to load DOX onto the “plain” ZnO NPs. Therapeutic agents could be loaded onto the ZnO NPs in different ways, including the electrostatic adsorption, hydrogen bonding, and zinc-mediated chelation [30]. Though the procedure of the DOX loading onto the ZnO NPs was simple and efficient, the exact mechanisms behind are not very clear, which may be involved in one or more aforementioned mechanisms. The dynamic light scattering (DLS) measurements showed that the average particle size of the plain ZnO NPs was 56.43 ± 12.05 nm and their zeta potential was + 24.22 ± 1.32 mV (Table 1). The loading of DOX onto the ZnO NPs didn’t significantly change the NPs’ particle size and zeta potential. Though the DOX-loaded ZnO NPs (ZnO/DOX) had a small particle size after the sonication or vortex (Fig. 1A), they tended to aggregate, as evidenced by the TEM images (Fig. 1C), which is unfavorable to drug delivery. Due to the strong UV interference between the zinc ions and DOX, the LC-MS/MS was used to determine the DOX content in ZnO/DOX. The drug loading efficiency of ZnO/DOX was around 16%, in consistent with the previous report [22].
Table 1. Particle size, zeta potential, polydispersity index (PDI), and drug (DOX) loading of the nanoparticles.
Name
|
Particle size (nm)
|
PDI
|
Zeta potential (mV)
|
Drug loading (%)
|
ZnO
|
56.43 ± 12.05
|
0.17 ± 0.06
|
24.22 ± 1.32
|
--
|
ZnO/DOX
|
57.30 ± 9.23
|
0.16 ± 0.02
|
21.40 ± 1.89
|
16.28
|
DPPG
|
343.77 ± 8.04
|
0.27 ± 0.01
|
-59.94 ± 3.20
|
--
|
ZnO/DPPG
|
47.05 ± 3.29
|
0.22 ± 0.01
|
-45.30 ± 1.90
|
--
|
ZnO/DPPG/PEG-PE
|
60.46 ± 6.12
|
0.13 ± 0.02
|
-16.98 ± 1.51
|
--
|
ZnO/DPPG/PEG-pp-PE
|
73.11 ± 10.76
|
0.20 ± 0.01
|
-19.16 ± 3.03
|
--
|
ZnO/DPPG/PEG-PE/DOX
|
60.11 ± 4.43
|
0.19 ± 0.03
|
-25.33 ± 0.54
|
10.33
|
ZnO/DPPG/PEG-pp-PE/DOX
|
83.25 ± 20.87
|
0.20 ± 0.02
|
-20.43 ± 1.06
|
11.49
|
The cellular uptake of ZnO/DOX was determined in DOX-sensitive (MDA-MB-231 and HeLa) cells and DOX-resistant (NCI/ADR-RES and MES-SA/Dx5) MDR cells. In the DOX-sensitive cells, the free DOX showed 2–3 times higher cellular uptake than that of the ZnO/DOX (Fig. S1), while in the DOX-resistant MDR cells, the ZnO/DOX showed the enhanced cellular uptake (> 2-folds than that of the free DOX). The confocal microscopy analysis also indicated that the free DOX had higher intracellular accumulation than that of the ZnO/DOX in the MDA-MB-231 cells, while the ZnO/DOX showed much stronger intracellular fluorescence than that of the free DOX in the NCI/ADR-RES cells (Fig. S2). To clarify the role of the ZnO NPs in the DOX uptake, the NCI/ADR-RES cells were pre-incubated with the ZnO NPs, followed by the DOX incubation. As shown in Fig. S3, the ZnO pre-incubation could not alter the cell internalization of the DOX, suggesting that the enhanced DOX uptake of ZnO/DOX was attributed to the DOX loading onto the ZnO NPs. It is well known that the used MDR cells overexpress the drug efflux pump, such as P-glycoprotein [31], lowering the intracellular concentration of various anticancer drugs, including DOX [22]. The data suggested that the ZnO NPs as a nanocarrier could not only enhance the cellular uptake but also attenuate the efflux of the loaded drug (DOX) in the MDR cancer cells.
The in vitro anticancer activities of the free DOX, ZnO NPs, and ZnO/DOX were evaluated by the MTT assay. The free DOX showed the highest cytotoxicity, while the ZnO NPs showed the lowest cytotoxicity in the DOX-sensitive tumor cells (Fig. S4). Here, the ZnO/DOX showed lower cytotoxicity than the free DOX, probably due to the insufficient cellular uptake (Fig. S1), low intracellular drug accumulation (Fig. S2), and sustained (slow) DOX release (from ZnO/DOX) (Fig. 1G). Due to the MDR, the cytotoxicity of the free DOX was remarkably decreased in the NCI/ADR-RES and MES-SA/Dx5 cells. The ZnO NPs showed cytotoxicity in both the sensitive and MDR cells, in consistent with our previous report [22]. The data indicated that the efflux pump might not be unable to pump the ZnO NPs out of the cells. Unsurprisingly, the ZnO/DOX showed the highest cytotoxicity against the MDR cells, suggesting that loading of DOX onto the ZnO NPs could effectively overcome the cancer cells’ MDR.
The drug penetration through the “3D” cancer cell spheroids was evaluated (Fig. S7). In the MDA-MB-231 spheroids, the free DOX showed good penetration (red fluorescence) and the ZnO/DOX just slightly increased the DOX penetration compared to the free DOX, as evidenced by the overall DOX uptake (area under curve) and penetration depth (the height of peak and the distance from the bottom to the top). However, in the NCI/ADR-RES spheroids, the penetration of the free DOX was significantly decreased due to the MDR. In contrast, the ZnO/DOX showed strong fluorescence even in the “core” of the spheroids, indicating that the ZnO NPs could overcome the drug resistance (efflux) in both the monolayer cells and their 3D spheroids.
These results suggested that the ZnO NPs could be an effective drug nanocarrier against the MDR cancer cells. However, the results also suggested that the ZnO NPs possessed some “unfavorable” properties, such as the low stability, biocompatibility, and specificity, usually resulting in the rapid systemic clearance and nonspecific biodistribution. For the successful in vivo drug delivery, the ZnO NPs need to be engineered to improve their biocompatibility, blood circulation time, and delivery specificity.
Synergistic anticancer effects of the ZnO/DOX NPs
Our previous study suggested that the ZnO NPs could induce the ROS production, leading to the death of cancer cells [22]. In this study, to further evaluate the ZnO-mediated cytotoxicity, the mitochondrial membrane potential was analyzed by the membrane-permeant cationic, fluorescent carbocyanine dye (JC-1) [32]. In the untreated cells with high mitochondrial transmembrane potential, JC-1 forms the J-aggregate complexes in the mitochondria and gives red fluorescence. In the treated/damaged cells, JC-1 remains in the monomeric forms in the cytosol as low mitochondrial transmembrane potential prevents its accumulation in the mitochondria and gives green fluorescence. We found that both the ZnO NPs and ZnO/DOX significantly decreased the mitochondrial membrane potential, as evidenced by the lowered Red/Green ratios (Fig. S5A) and decreased red fluorescence (and the increased green fluorescence) (Fig. S5B). Though DOX was reported to show mitochondrial toxicity, including the decreased membrane potential [33], the free DOX could not significantly alter the mitochondrial potential at the tested dose in the NCI/ADR-RES cells due to the MDR-induced low intracellular drug concentration (Fig. S2-3). Our data indicated that the ZnO NPs could exert cytotoxicity in cancer cells via the mitochondrial depolarization and ROS production. Interestingly, the NCI/ADR-RES cells seemed to be “tough” even after a short-time incubation with CCCP (Fig. S5A). But the mechanism needs to be identified.
C/EBP homologous protein (CHOP), a pro-apoptotic transcription factor encoded by the DDIT3 gene, plays an important role in endoplasmic reticulum (ER) stress-induced apoptosis [34]. Under normal physiological conditions, CHOP is ubiquitously present at a very low level. However, under overwhelming ER stress conditions, the CHOP expression rises sharply along with the activation of apoptotic pathways in a wide variety of cells. The increase in the CHOP level is an indicator of the ER stress. Therefore, the ER stress was examined by the determination of the CHOP level in the treated NCI/ADR-RES cells. As shown in Fig. S6, compared to the untreated group, no significant changes were observed in the levels of CHOP among the treatments, indicating that the ZnO NPs and DOX had the negligible effect on the ER stress in the MDR cells.
The results suggested that the ZnO NPs might enhance the DOX’s cytotoxicity in the MDR cells via the increased cellular uptake, intracellular accumulation, and zinc ion-induced mitochondrial dysfunction.
Preparation and characterization of the dual-responsive ZnO/DPPG/PEG-pp-PE/DOX NPs
PEGylation has been widely used to increase the NPs’ blood circulation and decrease their nonspecific interaction with the plasma proteins and mononuclear phagocyte system (MPS). By PEGylation, the NPs’ tumor targetability can be improved via the tumor’s EPR effect. On the other hand, however, PEGylation may impede the cell internalization of NPs, which may not be beneficial for intracellular drug delivery. Recently, in response to this dilemma, the stimuli-responsive NDDS has emerged as a smart tumor-targeted drug delivery system. In the tumor microenvironment, certain types of MMPs are upregulated and play important roles in cancer initiation, growth, and metastasis. They have been used as the robust tumor environmental stimuli for the stimuli-responsive NDDS [29]. In this study, we used the MMP2-sensitive polymer (PEG-pp-PE) to prevent the ZnO NPs from rapid systemic clearance and nonspecific distribution. In the tumor microenvironment, the peptide linker (pp) undergoes the MMP2-sensitive cleavage, resulting in the PEG deshielding and cellular uptake of the drug-loaded ZnO NPs (Scheme 1).
The synthesis of the MMP2-sensitive PEG-pp-PE was illustrated in Fig. S8A. The product was characterized by the TLC and 1H NMR (Fig. S8B-C), in consistent with our previous reports [35, 36]. To increase the surface lipophilicity of the ZnO NPs, the anionic lipid (DPPG) was first attached on the surface of the ZnO NPs via the electrostatic adsorption (referred to ZnO/DPPG), as evidenced by the change in the zeta potential from + 24.22 (ZnO) to -45.30 mV (ZnO/DPPG) (Table 1). We also found that the ZnO NPs failed to form a stable suspension in the chloroform and quickly settled to the bottom of the bottle, while after the DPPG modification, the ZnO/DPPG were evenly dispersed in chloroform, probably due to the increased stability (Fig. 1E).
Then, the ZnO/DPPG were PEGylated by the MMP2-sensitive PEG-pp-PE or non-sensitive PEG-PE through the emulsification and solvent evaporation method to obtain the ZnO/DPPG/PEG-pp-PE and ZnO/DPPG/PEG-PE. The DOX was readily loaded to the ZnO based NPs via the simple incubation with the doxorubicin hydrochloride solution [22] to form the ZnO/DPPG/PEG-pp-PE/DOX and ZnO/DPPG/PEG-PE/DOX, respectively. The hydrodynamic particle size and zeta potential of the ZnO NPs and their formulations were measured by the DLS in Hank’s balanced salt solution (HBSS) (Table 1). The DPPG dispersion exhibited a large particle size probably due to the formation of the large liposome-like structure in water, while the ZnO NPs and their formulations had the particle sizes in the range of ~ 50–100 nm. Without the surface modification, the ZnO NPs were positively charged, and the DOX loading didn’t significantly influence the NPs’ zeta potential and particle sizes. In contrast, the ZnO/DPPG based NPs were negatively charged due to the anionic lipid, DPPG. The PEGylation slightly increased the ZnO/DPPG NPs’ particle sizes (Fig. 1B) but lowered their negative charge from ~ -45mV to ~ -20mV, due to the PEG’s steric hindrance (Table 1). The drug loading efficiency in the ZnO/DPPG/PEG-pp-PE/DOX and ZnO/DPPG/PEG-PE/DOX were 11.49% and 10.33% respectively, which was determined by the LC-MS/MS.
Stability, sensitivity and drug release of the ZnO/DPPG/PEG-pp-PE/DOX NPs
The TEM images indicated that the ZnO/DPPG/PEG-pp-PE/DOX were well dispersed in HBSS with a spherical morphology (Fig. 1D), while the ZnO/DOX showed an aggregation propensity (Fig. 1C). The ZnO/DPPG/PEG-pp-PE/DOX exhibited excellent NP stability in both the HBSS (pH 7.4) and HBSS containing 10% FBS for 72 h (Fig. 1F and S9). In our previous study, we demonstrated that the PEG-pp-PE was MMP2-sensitive and could be cleaved by MMP2 within 4 h [36]. Here, to better understand the influence of the MMP2 sensitivity on the NP stability, we prolonged the MMP2 incubation time to 12 h to ensure the complete cleavage although the 1 h MMP2 incubation was sufficient to trigger the efficient cellular uptake (see Fig. 2). After the 12 h MMP2 incubation, the lose aggregates were observed in the ZnO/DPPG/PEG-pp-PE/DOX group, while the ZnO/DPPG/PEG-PE/DOX remained no change (Fig. S10A). The data suggested that MMP2 could cleave PEG-pp-PE and remove the PEG shell from the NPs, resulting in the decreased NP stability in the aqueous environment. However, most of the MMP2-treated NPs were still in the nanometer range (< 200nm) upon mild agitation (Fig. S10B).
The drug release patterns of the ZnO/DOX and ZnO/DPPG/PEG-pp-PE/DOX were studied by the dialysis method [22]. As shown in Fig. 1G, the ZnO/DOX exhibited a slow drug release rate at pH 7.4 with a < 50% DOX release at 24 h, while its drug release rate was dramatically increased at pH 5.0 with a > 90% DOX release at 10 h, which was similar to the release pattern of the free DOX. The data indicated that the DOX release from the ZnO NPs was pH-dependent due to the acidic pH-induced dissociation/dissolution of the ZnO NPs. The DPPG modification and PEGylation further slowed down the DOX release (from ZnO/DPPG/PEG-pp-PE/DOX) compared to the ZnO/DOX (Fig. 1H vs. 1G). However, the ZnO/DPPG/PEG-pp-PE/DOX showed a < 25% DOX release at pH 7.4 and an around 70% drug release at pH 5.0 after 48 h dialysis (Fig. 1H), indicating that these surface modifications didn’t significantly influence the ZnO NPs’ pH sensitivity. We also found that both the MMP2 and human serum albumin (HSA) incubation didn’t significantly increase the DOX release from ZnO/DPPG/PEG-pp-PE/DOX (with a less than 20% DOX release after 48 h incubation). Although the MMP2-mediated PEG-pp-PE cleavage “released” the ZnO/DPPG/DOX resulting in the slight aggregation (Fig. S10A), the loaded DOX remained loaded on the surface of the ZnO NPs without drug release/leakage. The high drug retention in the ZnO NPs even after the MMP2-mediated cleavage would ensure the drug’s cellular uptake in the tumor microenvironment. Here, we chose the water-soluble doxorubicin hydrochloride salt instead of the insoluble DOX base to facilitate the DOX loading onto the surface of the ZnO NPs rather than the drug entrapment in the lipid (DPPG and PE) layer of the NPs, by which the DOX’s pH-dependent release was secured.
Cellular uptake and penetration of the ZnO/DPPG/PEG-pp-PE/DOX NPs
The cellular uptake and penetration of the ZnO/DPPG/PEG-pp-PE/DOX were evaluated on the MDR cancer cells (NCI/ADR-RES and MES-SA/Dx5) and their 3D spheroids. After 1 h cell incubation, the ZnO/DOX exhibited the highest cellular uptake in all cancer cells probably due to their positive charge-mediated uptake and NP-mediated efflux inhibition (Fig. 2). The ZnO/DPPG/DOX decreased DOX’s cellular uptake compared to the ZnO/DOX, probably due to the charge neutralization by DPPG. The ZnO/DPPG/PEG-PE/DOX and ZnO/DPPG/PEG-pp-PE/DOX exhibited lower cellular uptake, which might be caused by the PEG’s steric hindrance. To study the impact of the MMP2-mediated cleavage on cellular uptake, the ZnO/DPPG/PEG-PE/DOX and ZnO/DPPG/PEG-pp-PE/DOX were preincubated with MMP2 or HAS for 1 h before incubating with the cells. No significant increase in the cellular uptake was observed in the ZnO/DPPG/PEG-PE/DOX + MMP2 treated cells, while the ZnO/DPPG/PEG-pp-PE/DOX + MMP2 showed higher cellular uptake, indicating that the MMP2-mediated PEG-deshielding could enhance NPs’ cellular uptake. It was worth noting that, compared with the ZnO/DPPG/PEG-PE/DOX and ZnO/DPPG/PEG-pp-PE/DOX, there was just a slight change in the cellular uptake of the ZnO/DPPG/PEG-PE/DOX + HSA and ZnO/DPPG/PEG-pp-PE/DOX + HSA, probably due to the HSA-mediated cell internalization [37]. In the MES-SA/Dx5 cells, the similar MMP2-mediated enhanced cellular uptake was observed (Fig. S11A). We also noticed that the cellular uptake of the ZnO/DPPG/PEG-pp-PE/DOX + MMP2 was even higher than that of the ZnO/DPPG/DOX, probably because of the MES-SA/Dx5 cells’ different response to the negatively charged ZnO/DPPG/DOX. The confocal microscopy results (Fig. 2B and S11B) confirmed the flow cytometry data that the cellular uptake of the ZnO/DPPG/PEG-pp-PE/DOX was significantly enhanced after the MMP2 pre-incubation. After cell internalization, the DOX (red) was co-localized with the Hoechst (blue), which showed pink, indicating the drug accumulation in the cell nuclei.
Cancer cells cultured as the 3D cell spheroids present the similar characteristics to those of the in vivo tumor, including the morphology, growth kinetics, gene expression, drug response, etc. [38]. It has been also shown that the cell spheroids exhibit the drug resistance more relevant to the in vivo tumor as compared to the cells grown in the monolayer [39]. Thus, the NCI/ADR-RES cell spheroids were used to further evaluate the anti-MDR effect of the DOX-loaded NPs. The penetration of the DOX-loaded NPs through the cell spheroids was evaluated by confocal microscopy (Fig. 3). After 4 h incubation, both the ZnO/DPPG/PEG-PE/DOX and ZnO/DPPG/PEG-pp-PE/DOX showed the limited penetration in the cell spheroids, due to the spheroid’s 3D architecture, cells’ drug efflux/resistance, and NPs’ PEGylation (uptake inhibition) [22, 31]. After the MMP2 pre-incubation, the penetration ability of the ZnO/DPPG/PEG-PE/DOX + MMP2 did not significantly increase, while the ZnO/DPPG/PEG-pp-PE/DOX + MMP2 showed the enhanced spheroid penetration, as evidenced by the strong red fluorescence in the core of the spheroids.
These results suggested that, in response to the MMP2, the dual-responsive ZnO/DPPG/PEG-pp-PE/DOX could deshield the PEG shell and the exposed ZnO/DPPG/DOX NPs could enhance the tissue (spheroid) penetration and cellular uptake. Inside the cells, the ZnO was dissociated/dissolved at endosomal acidic pH, resulting in the DOX release. By this design, the efflux-mediated DOX resistance would be overcome.
In vitro cytotoxicity of the ZnO/DPPG/PEG-pp-PE/DOX NPs
In order to verify whether the enhanced cellular uptake and penetration of the ZnO/DPPG/PEG-pp-PE/DOX could lead to the increased anticancer activity, the in vitro cytotoxicity of the ZnO-based NPs on the MDR cancer cell monolayers and spheroids was evaluated by the MTT assay and CellTiter-Blue® cell viability assay. In the cell monolayers, the ZnO/DOX and ZnO/DPPG/DOX showed the highest cytotoxicity against both MDR tumor cells (NCI/ADR-RES and MES-SA/Dx5) (Fig. 4A and S13). The cytotoxicity of the ZnO/DPPG/PEG-PE/DOX didn’t show the noticeable change after the MMP2 pre-incubation, while the cytotoxicity of the ZnO/DPPG/PEG-pp-PE/DOX was significantly increased. Here, the ZnO/DPPG/DOX and ZnO/DOX showed the similar cytotoxicity although the ZnO/DOX had higher cellular uptake than ZnO/DPPG/DOX, which may be attributed to the prolonged cell incubation time. Besides, we found that without the MMP2 pre-incubation, the ZnO/DPPG/PEG-pp-PE/DOX also exhibited higher cytotoxicity than the ZnO/DPPG/PEG-PE/DOX, probably because that the endogenous MMP2 secreted by the cancer cells cleaved the PEG-pp-PE, leading to the increased cellular uptake [40].
To further investigate the anticancer activity of the DOX-loaded NPs, the morphology and cell viability of the NCI/ADR-RES spheroids were determined (Fig. S12 and 4B). The free DOX and ZnO NPs showed mild toxicity individually while the DOX-loaded ZnO NPs, including the ZnO/DOX and ZnO/DPPG/DOX, exhibited the most significant toxic effects on the cancer cells including the highest cytotoxicity and the damaged spheroid architecture, probably due to the synergistic effect of the DOX and ZnO (Fig. S4 and [22]). After the ZnO/DOX or ZnO/DPPG/DOX treatment, a blurry edge was observed, which was due to a significant shedding of cells or cell debris from the spheroids (Fig. S12). This was probably because of the rapid uptake (Fig. 2) and strong toxic effects (Fig. 4) of the ZnO/DOX or ZnO/DPPG/DOX on the outer layers of the cell spheroids. Without the MMP2 pre-treatment, all PEGylated NPs showed low cytotoxicity (Fig. 4) and negligible influence on the spheroid morphology (Fig. S12). The MMP2 pre-treatment significantly increased the cytotoxicity of the PEG-pp-PE modified NPs (ZnO/DPPG/PEG-pp-PE/DOX + MMP2) rather than the PEG-PE modified NPs (ZnO/DPPG/PEG-PE/DOX) (Fig. 4).
These results suggested that after loading to the dual-responsive ZnO/DPPG/PEG-pp-PE NPs, the efflux-induced DOX resistance could be overcome in the MDR cancer cells and their 3D spheroids, resulting in the enhanced anticancer activity.
In vivo biodistribution of the ZnO/DPPG/PEG-pp-PE/DOX NPs
The in vivo biodistribution and tumor targeting of the dual-responsive NPs were evaluated in the NCI/ADR-RES xenograft mouse model, by the live animal imaging. Due to the low tissue penetration and high absorption and emission interference of the DOX fluorescence, in this study, the Cy5.5-labeled NPs were prepared for the animal imaging (Fig. 5A). Within the 24 h upon intravenous (i.v.) injection, the strong fluorescence was observed in the liver and kidney in the ZnO/DOX - treated mice while no fluorescence (v.s. background) was detected in the tumor site. In contrast, the fluorescence of the liver and kidney in the ZnO/DPPG/PEG-pp-PE/DOX - treated mice was relatively low, while the fluorescence signal in the tumor was detected 2 h after intravenous injection. At 24 h post administration, the mice were sacrificed, and the major organs and tumor were harvested for the ex vivo fluorescence measurement (Fig. 5B). In the ZnO/DOX - treated mice, the fluorescence could be observed in all major organs with the strong fluorescence in liver and kidney while the fluorescence in the tumor was negligible. In the ZnO/DPPG/PEG-pp-PE/DOX - treated mice, the fluorescence intensity was low in the excised organs except kidney, while the fluorescence in the tumor tissue was strong. These results suggested that, without PEGylation, the positively charged ZnO/DOX would be most likely opsonized and captured by the mononuclear phagocyte system (MPS), including liver and kidney, before reaching the tumor site. In contrast, the PEGylated dual-responsive ZnO/DPPG/PEG-pp-PE/DOX NPs could effectively minimize the opsonization, thus prolonging the blood circulation time and facilitating the EPR effect-mediated tumor accumulation. In addition, the MMP2-mediated cellular uptake secured the tumor retention of the ZnO/DPPG/PEG-pp-PE/DOX.
In vivo anticancer activity of the ZnO/DPPG/PEG-pp-PE/DOX NPs
The anticancer activity of the DOX-loaded NPs was evaluated after the i.v. injection at 5mg/kg DOX in the NCI/ADR-RES tumor-bearing mice. As shown in Fig. 6A, the tumor volume of the saline-treated group dramatically increased over 14 days. Compared to the saline group, the tumor growth in the free DOX and ZnO/DOX treated mice was just slightly inhibited, indicating their failure in the in vivo cancer treatment although they could efficiently kill cancer cells in vitro (Fig. 4). The PEGylated NPs significantly inhibited the tumor growth, due to their long blood circulation and tumor’s EPR effect. Compared to the ZnO/DPPG/PEG-PE/DOX, the dual-responsive ZnO/DPPG/PEG-pp-PE/DOX displayed higher anticancer activity. The tumor growth inhibition data were well consistent with the tumors’ pictures and weights (Fig. 6B). The TUNEL assay showed that more tumor cells underwent apoptosis/necrosis in the ZnO/DPPG/PEG-pp-PE/DOX treated mice, compared to those of other treatments, which was confirmed by the H&E staining (Fig. 6C). We observed that, unlike the in vitro data, the PEGyated NPs (ZnO/DPPG/PEG-PE/DOX and ZnO/DPPG/PEG-pp-PE/DOX) exhibited a strong anticancer activity than the ZnO/DOX. The data confirmed that the ZnO/DOX was effective only in the cell cultures, whereas our dual-responsive delivery strategy could improve the in vivo biocompatibility, biodistribution, tumor targeting, and anticancer activity of the ZnO/DOX NPs.
During the study, no significant changes in the mouse body weight were observed (Fig. 7A). To study the in vivo toxicity of the treatments, the alanine aminotransferase (ALT), aspartate aminotransferase (AST), the serum creatinine (CREA), and the white blood cells (WBT) were measured (Fig. 7B). No significant changes were observed in the levels of AST and CREA among the treatments. However, compared with other treatments, the ZnO/DOX increased the ALT level and decreased the WBC. The free DOX also showed the increased toxicity though it was not statistically significant (Fig. 7B). The histological staining of the major organs including heart, liver, spleen, lung, and kidney was shown in Fig. 7C. Except for the ZnO/DOX group, other treatments didn’t apparently damage the organs compared to the saline group. In the ZnO/DOX group, the inflammatory cell infiltration in the liver and the necrosis of epithelial cells in kidney tubules were found. The toxicity data indicated that the i.v. injection of the ZnO/DOX could result in serious hepatic and renal toxicity, and other potential side effects, which was consistent with the previous report [41]. This might be caused by the ZnO/DOX’s nonspecific bio-interactions and capture by the liver and kidney. In contrast, the prepared dual-responsive ZnO/DPPG/PEG-pp-PE/DOX NPs significantly improved the biocompatibility and safety of the ZnO/DOX.