Synthesis and characterization of nanoparticles
Nanoparticles with the material of CS-ADH-Rh-LA (NC-NPs and C-NPs representing the non-crosslinking and crosslinking nanoparticles) were prepared by simple sonication with the size ranging from 158.4 to 192.4 nm (Table 1). Although the Rh served as the hydrophobic part, the sizes of both NC-NPs and C-NPs increased with the increase of Rh to CS-ADH ratio, probably owing to the steric hindrance effect of Rh molecules. CMC value of NC-NPs with different ratios of Rh to CS-ADH was ranged from 33.74 to 66.54 µg/mL, which indicated excellent self-aggregation ability and dilution stability of NC-NPs (Table 1). Compared with NC-NPs, C-NPs had small size, more spherical shape and good dispersion (Fig. 1A and 1B), owing to their improved steadiness after crosslinking [30]. While both NC-NPs and C-NPs exhibited good stability with zeta potential of -28.9 mV and − 30.9 mV, respectively (Table 1). In addition, the 1H-NMR, 13C-NMR and FT-IR results of CS-ADH-Rh-LA were shown in Fig. S2.
Table 1
Characteristics of CS-Rh-LA nanoparticles. Values are listed as Mean ± SD, n = 3
|
NC-NPs
|
C-NPs
|
Sample
|
Rh DS (%)
|
Diameter (nm)
|
PDI
|
ζ (mV)
|
CMC (µg/mL)
|
Diameter (nm)
|
PDI
|
ζ (mV)
|
CS-Rh-LA1
|
0.93
|
165.7 ± 4.23
|
0.153 ± 0.015
|
-15.6
|
66.54
|
138.5 ± 3.50
|
0.128 ± 0.018
|
-25.7
|
CS-Rh-LA2
|
2.69
|
180.6 ± 3.90
|
0.166 ± 0.029
|
-18.8
|
47.47
|
163.8 ± 3.09
|
0.122 ± 0.022
|
-26.8
|
CS-Rh-LA3
|
3.15
|
184.4 ± 2.50
|
0.100 ± 0.014
|
-21.0
|
33.74
|
178.3 ± 2.65
|
0.0913 ± 0.008
|
-28.9
|
Stability of nanoparticles
The stability of C-NPs was also evaluated when exposed to different concentrations of DTT solution (Fig. 1E). According to the result, the size of C-NPs increased from about 180.8 nm to 272.5 nm, 401.6 nm and 715.0 nm after 6, 12 and 24 h of incubation with 20 mM DTT solution, respectively, manifesting C-NPs swelled slightly and aggregated with crushed structure after 24 h incubation. Therefore, the monodispersed nanospheres changed into irregular stacks after 24 h as observed by TEM (Fig. 1C and 1D). However, there was negligible change in size of C-NPs incubated with 20 µM DTT solution (Fig. S3). In addition, there was little change in size of NC-NPs after incubating with DTT solutions. Further, in vitro stability was evaluated after incubating with PBS (pH7.4, 10 mM), NaCl and complete medium with 10% FBS for 24 h (Fig. 1F). In both PBS and NaCl solution, NC-NPs showed a decrease in size, while C-NPs maintained favorable colloidal stability. Particularly, the stability of C-NPs was better than NC-NPs in 10% FBS solution within 6 days (Fig. S4). In addition, to estimate blood stability, the hemolysis rate of C-NPs was studied (Fig. 1G). Although the hemolysis rate was increased with the concentration of C-NPs but was below 5% in all contents (0.1 to 1 mg/mL), showing good biocompatibility of the synthesized polymers. Therefore, C-NPs possessed superior colloidal stability after crosslinking in normal physiological environment or blood, and disassembled quickly under simulative tumor microenvironment, guaranteeing their successful delivery.
Drug loading and release assay
To realize the combined therapy, DTX was encapsulated in C-NPs through sonication-dialysis method, and the effect of DTX to C-NPs ratio on drug loading was studied as shown in Table 2. As the raising of charge ratio (1:10 ~ 5:10), C-NPs could carry more DTX with the drug loading (DL%) rose from 2.50–12.05%. While corresponding encapsulate efficiency (EE%) increased from 23.93–35.73% as the ratio increased from 1:10 to 3:10, but at last reduced to 28.25% at 5:10 ratio, which could be explained by the breakage of hydrophobic-hydrophilic balance for superfluous addition of DTX. Besides, the size increased as the proportion of DTX increases, but the zeta potential showed the highest absolute value only at the ratio of 3:10. Over all the considerations, the 3:10 ratio of DTX to C-NPs was selected for further studies. After crosslinking, the in vitro release profile of DTX/C-NPs was evaluated in PBS solution containing 20 µM and 20 mM DTT, respectively (Fig. 1H). The cumulative release of DTX in PBS containing 20 mM DTT was more rapid in the first 12 h (nearly 80%) than the PBS containing 20 µM DTT and PBS without DTT, and reached to approximately 93% compared to 56% and 53% for 20 µM DTT or PBS group, respectively, at 72 h. Therefore, the drug delivery system could self-dissociate quickly and release the drug intelligently as the change of environment.
Table 2
Characteristics of DTX/CS-ADH-Rh-LA nanoparticles. All values are presented as Mean ± SD (n = 3)
Sample
|
DTX / polymer
|
DL (%)
|
EE (%)
|
Diameter (nm)
|
ζ (mV)
|
CS-ADH-Rh-LA
|
1 : 10
|
2.50 ± 0.18
|
23.93 ± 2.26
|
193.5 ± 1.2
|
-24.2
|
2 : 10
|
5.71 ± 0.15
|
31.75 ± 2.11
|
201.7 ± 2.2
|
-25.2
|
3 : 10
|
9.57 ± 0.38
|
35.73 ± 1.97
|
231.7 ± 3.9
|
-30.7
|
5 : 10
|
12.05 ± 0.25
|
28.25 ± 1.37
|
254.0 ± 2.3
|
-28.9
|
Cellular uptake process
FCM and CLSM were used to analyze and observe the internalization of coumarin 6 (C6)-loaded C-NPs prepared by the same method of DTX/C-NPs. The fluorescent-labeled cells were screening out followed by compiling the statistical data in histogram form (Fig. 2A). It was indicated that the C-NPs could internalize to A549 cells in time-dependent way as fluorescent signal became the strongest after 4 h of incubation. The signals for free coumarin group appeared in the same way, but the fluorescence intensity was much weaker than the C-NPs group. Meanwhile, the cellular uptake mechanism was studied using CLSM, as shown in Fig. 2C. In this part, the C6-loaded C-NPs were compared with free C6 and CS blocking group (A549 cells were treated with CS solutions in advance) at different time points. Similar to FCM result, the fluorescence of C6/C-NPs was fiercer than the C6 free group, while no signal was detected for the control group. The internalizing capacity of CS blocking group was between the control and free C6 group, manifesting successful blocking process of CD44 receptors and confirming the active targeting-mediated internalization of C-NPs. For H1299 cells, the fluorescence intensity was much weaker than the A549 cells on even ground (shown in Fig. 2D), confirming the abundance of CD44 receptors on A549 cells.
Further, the exocytosis-dependent transcellular transport of C6 loaded C-NPs was measured among disparate batches of A549 cells by means of simulation. According to Fig. S5, fluorescence intensity of C6 was high and visible in cells on coverslips (I) and (II), which indicated that certain C6 taken up at the first step was able to secrete into the new medium and then be taken as well as absorbing by cells on coverslip (II). However, the coverslip (III) showed scarcely fluorescent signal, which might be attributed to the reduction of fluorescent material. Collectively, C-NPs were actively taken up by cancer cells due to the overexpression of CD44 receptor, and the intake increased with time. Besides, the C-NPs were entered the cells via endocytosis and delivered to the surrounding cells through exocytosis, which might lay the foundation for active penetration, subsequently.
Cytotoxicity and apoptosis
The C-NPs- and DTX/C-NPs-induced cytotoxicity and apoptosis to A549 cells were evaluated. First, CCK-8 method was applied to estimate cytotoxicity of C-NPs to A549 cells. The cells were incubated with C-NPs at different concentration gradient of Rh, and ultrasound (1.2 W/cm2) was applied on those samples for 0, 1, 3 and 5 min (the groups were named as darkness, SDT-1, SDT-3 and SDT-5), respectively. According to the results, the C-NPs exhibited negligible cytotoxicity in dark environment at 0.1 µg/mL (Fig. 3A). While the dark cytotoxicity increased with the rise of Rh concentration and showed similar trend with those of SDT groups at 32 µg/mL concentration. However, the SDT groups exhibited significantly enhanced cytotoxicity (p < 0.01) in an ultrasonic time-dependent way. The IC50 value was 1.061, 0.5664, 0.5049 and 0.2959 µg/mL for the dark, SDT-1 min, SDT-3 min and SDT-5 min groups, respectively. As manifested by Fig. 3B, C-NPs and DTX/C-NPs showed remarkable cell killing ability at low concentrations compared with free Rh under same environment, which probably attributed to the active targeting of the C-NPs. Besides, DTX/C-NPs manifested stronger cell killing effect after SDT treatment especially at low concentrations.
Further, the synergistic cytotoxicity of DTX/C-NPs against A549 and H1299 cells was studied (Fig. 3C and 3D). For A549 cells, DTX/C-NPs showed excellent cell killing effect even at low concentration of 10− 3 µg/mL in darkness with cell viability of 64.3% compared with H1299 cells of 93.8%, not to mention the SDT-treated groups. The results not only indicated the preferably active targeting of CS to CD44 receptors expressed on A549 cells rather than H1299 cells, but also the reduced cytotoxicity of DTX/C-NPs than free DTX solution (Fig. S6). Further, it could be deduced that cytotoxicity of C-NPs to was nearly doubled after encapsulating DTX with/without SDT treatment to A549 cells. In addition, the cell viability of CS-ADH-LA polymer was above 80% at all concentrations, exhibiting good safety (Fig. S7). Under the comprehensive consideration of effective cytotoxicity and shorter ultrasonic time, A549 cells and SDT for 3 min were applied for further experiments. Subsequently, the efficacy of DTX/C-NPs with SDT was evaluated through a live/dead-cell staining study with calcein-AM (ex/em: 490/515 nm) together with propidium iodide (PI) (ex/em: 517/617 nm) agents (Fig. 3E). The red fluorescence (dead cells) emerged and became stronger after incubating with DTX/C-NPs after SDT treatment compared with the control group, demonstrating their favorable ROS generating property. However, scarcely any red fluorescence was observed for free Rh group than Rh + SDT group, revealing good sonodynamic efficiency after SDT treatment for free Rh.
Merely ultrasound or Rh induced trivial apoptosis and necrosis to A549 cells (mortality ratio < 1%) according to the cell apoptosis result by FCM, and the apoptosis rate of SDT control group and CS-ADH-LA was similar (Fig. 3F). Besides, the mortality ratio of free Rh with SDT (6.9% of cell apoptosis rate) was enhanced than the control group, but far from DTX/C-NPs with SDT group (10.0% and 2.9% of cell apoptosis and necrotic rate). In addition, the stronger mortality ratio of Rh with SDT group than DTX/C-NPs group highlighted the importance of Rh as well as SDT treatment in tumor suppression in A549 cells, and the SDT induced distinct karyopyknosis and karyolysis compared with the group without SDT treatment at the same time point after culturing with DTX/C-NPs (Fig. 3G).
ROS detection during SDT process
The generation of ROS was considered crucial for the apoptosis and cellular components destruction of cancer cells [31], attributing to the addition of Rh with ultrasonic therapy and the consumption of GSH by NPs. The formation of intracellular ROS was detected in A549 cells using CLSM and FCM after different incubation times. Firstly, after 4 h, the Rh + SDT group manifested obvious green fluorescence compared with Rh group, and the fluorescence of NAC (ROS scavenger) and control group was negligible (Fig. S8). However, it was worth noting that Rh + SDT group and C-NPs + SDT group showed similar ROS producing capacity (Fig. S8 and Fig. 4A), which probably owing to the short incubation time that limited the ROS production. Then, to prove the correlation between effective SDT and intact structure of Rh, the singlet oxygen contents were detected with different agents using 9, 10-dimethylanthracene (DMA) after sonicating for 4 h. However, the peaks of Rh and C-NPs decreased fiercely compared with Ce6, EMO and the control group, and C-NPs generated even more singlet oxygen than C-NPs (Fig. 4B). Next, we observed the fluorescence image of ROS with different concentrations of C-NPs or various incubation times to further verify the above hypothesis (Fig. 4C and Fig. 4D). To our anticipation, ROS was produced in a time and concentration dependent way. Nevertheless, fluorescence weakened at 5 µg/mL of C-NPs after 24 h incubation, which might be attributed to the apoptosis of A549 cells at such high concentration.
Detection of MMP
The change of MMP was the indication of destruction of Adenosine Triphosphate (ATP) producing process via utilizing the proton electrochemical gradient potential across the mitochondrial membrane [32]. We used jc-1 assay kit to determine the variation of MMP by FCM, and the y-axis and x-axis represented MMP in normal cells (red fluorescence) and abnormal cells (green fluorescence), respectively. The graph was divided into four parts, in which Q1 or Q4 represented pure fluorescence positive for red or green, while Q2 or Q3 as double positive or double negative, respectively. Control groups with/without SDT were located in Q1 zone predominantly, manifesting the formulation of jc-1 aggregates with high MMP level. However, the MMP was decreased in Rh + SDT and C-NPs + SDT groups, indicating the rupture of mitochondrial membrane and the dis-formable of jc-1 aggregates, and the destruction of mitochondrial membrane was aggravated with SDT time (Fig. 4E). According to the result, the green fluorescent ratio (sum of Q2 and Q4 zones) was 0.9% and 3.6% for control and control + SDT group, respectively, while increased to 30.6% and 49.4% for Rh and DTX/C-NPs groups after ultrasonicating for 5 min. Therefore, ultrasound could damage the mitochondrial membrane, leading to cellular apoptosis in a time dependent way with the help of SSs.
DTX/C-NPs induces cell cycle arrest in A549 cells by facilitating microtubule polymerization
In order to study the growth inhibition effect of DTX/C-NPs to A549 cells, microtubule morphology and cell cycle were observed and analyzed. According to the result, the microtubule (marked by α-tubulin protein) had clear morphology and ran through the structure of A549 cells. However, the microtubule disappeared and formulated polymers on certain parts of A549 cells after culturing with DTX or DTX/C-NPs, manifesting the cytotoxicity and inhibition of cell division by DTX. Moreover, the SDT treatment facilitated the microtubule polymerization to a certain extent, though weak, as the images of control and Rh + SDT showed brighter red fluorescence in some parts of the cell (Fig. 5A). In addition, we investigated the influence of DTX/C-NPs to cell cycle arrest using a DNA detection kit with PI staining (Fig. 5C). Compared to control group, the percentage of G2/M increased from 21.06–23.20%, 25.76%, 60.88% and 62.01% for Rh + SDT, control + SDT, DTX and DTX/C-NPs + SDT groups, respectively. Therefore, A549 cells could be arrested in G2/M phase by promoting microtubule polymerization or preventing microtubule depolymerization with the help of DTX or DTX/C-NPs.
Western blot result
The apoptosis related proteins were measured via western blot assay (Fig. 5B). Since the apoptosis was related to poly ADP-ribose polymerase (PARP) cleavage, typical hallmarks of caspase-3 and activated caspase-3 that were affirmed during the apoptosis were detected. Compared with the control group, DTX/C-NPs + SDT group expressed minimum amount of caspase-3 protein, but the maximum amount of cleaved caspase-3 protein. Additionally, the expression of MMP9 was also determined, which was regarded as an enzyme with the affinity to zinc, and could degrade the extracellular matrix as well as promote the invasion of cancer cells [33]. Besides, the expression of MMP9 decreased after incubating with DTX/C-NPs with/without SDT than control and Rh treated groups, proving the good anti-metastasis activity of DTX/C-NPs and SDT. However, there was little changes for VEGFA protein expression between different groups, manifesting the minimum effect of Rh in the inhibition of tumor angiogenesis with DTX/C-NPs or SDT at such a low concentration.
Subcellular localization and distribution
Subcellular localization and distribution were also studied by using C6/C-NPs. After staining with Lyso-Track Red, Mito-Tracker Red CMXRos and Golgi-Tracker Red mark for marking the lysosomes, mitochondria and Golgi apparatus, respectively, the cells were observed at different time points (Fig. 6). The results showed that only few signals of C6 colocalized with lysosomes and mitochondria, and their max Pearson correlation coefficients were 0.56 or 0.51 after incubating for 3 h, respectively (Fig. 6A, 6B, 6 Da and b). On the contrary, C-NPs rapidly colocalized with Golgi apparatus after the NPs was internalized into the cells for 1 h, and the Pearson correlation coefficient even reached to 0.92 (Fig. 6C and Fig. 6Dc). Therefore, it was preliminary speculated that the internalized C-NPs were transported to the Golgi apparatus within 1 h, and were gradually transferred to lysosome for further enzymolysis between 1 h and 3 h after the colocalization to Golgi membrane reached saturation. However, for mitochondria, the Pearson correlation coefficient had little difference for 1 h, 3 h and 6 h, but was significantly declined at 12 h, which might be attributed to the rupture of mitochondrial membrane after culturing for 12 h.
Destroy of Golgi structure
Since the Golgi apparatus was regarded as hub for sorting and trafficking of intracellular proteins, and could support cellular trafficking via balancing the of protein and membrane [34], the change in Golgi structures in connection with cancer disease was observed via immunofluorescent staining method. In our work, the stability of Golgi tethering protein GM130 (Golgi matrix protein) was investigated. The GM130 in DTX/C-NPs + SDT group showed obvious depolymerization as the number of red small spots were increased (Fig. 7A). Meanwhile, the small spots were counted and their average was calculated after incubating for 12 h (Fig. 7B). According to the result, the number of GM130 protein were in the sequence of Rh ≈ Control (+/-SDT) < DTX/C-NPs ≈ Rh (+ SDT) < DTX/C-NPs (+ SDT), manifesting the increasing disruption of Golgi structures among the groups that would lead to the increase of migration, invasion and poor prognosis of lung cancer [35].
Inhibition of cell migration and invasion
Since the migratory as well as invasive abilities are crucial to evaluate the metastatic cascade of cancer cells, the wound-healing assay together with the transwell invasion assay were performed in order to evaluate the migration and invasive ability on A549 cells with different solutions at their IC50 concentrations. According to the scratch test, the wound closure of control group could reach to 71.5% after 24 h, and changed to 67.1% after the SDT treatment. However, the rate was increased (75.5%) in C-NPs solution, but significantly reduced (34.9%) after SDT treatment, proving the inefficient migration inhibition of C-NPs to A549 cells. Besides, the values decreased to 62.6%, 52.7%, 32.9%, 27.5% and 15.8% for Rh, Rh + SDT, DTX, and DTX/C-NPs and DTX/C-NPs + SDT groups, respectively, manifesting the good anti-metastatic effect of SDT and DTX (include DTX/C-NPs) (Fig. 7C and 7D). We further investigated the invasive ability on the basis of migration assay, which showed a similar trend (Fig. 7E and 7F). The cell number was counted and the average cell number across the Matrigel was 227.4 ± 25.9 and 225.6 ± 25.6 for control and control + SDT groups. The C-NPs group showed similar invasion ability like the control one with cell number of 231.4 ± 23.8, but the value decreased, significantly, after SDT to 106.2 ± 17.4. As for DTX/C-NPs, the cell number showed persisting declination to 71.2 ± 9.4 and 33 ± 5.4 for DTX/C-NPs without and with SDT, respectively. Consequently, the SDT and DTX/C-NPs exhibited important roles in inhibiting cellular migration and invasion.
Biodistribution of Dir/C-NPs
In vivo imaging was conducted after injecting Dir (1,1-dioctadecyl-3,3,3,3-tetramethy lindotricarbocyaineiodide)-loaded C-NPs or free Dir solution intravenously to the A549 tumor bearing nude mice at the time points of 1, 4, 12 and 24xh, respectively. The whole mice bodies were scanned, photographed, and analyzed by the Caliper IVIS Lumina II system (Fig. 8A). At 1 h, the fluorescence signals of Dir reached to the maximum for both Dir/C-NPs and free Dir groups, manifesting the active targeting ability of C-NPs and quick distribution of Dir. However, the Dir signals weakened quickly in the free Dir group than Dir/C-NPs with the passage of time, owing to the long circulating ability of CS and active targeting of the nanoparticles compared with the rapid metabolism of free drug. After intravenous injection for 24 h, mice were executed and dissected to acquire their main organs for fluorometric analysis (Fig. 8B and 8C). Compared to the results of in vivo biodistribution images at 24 h, C-NPs exhibited strong fluorescence intensity after dissection especially in tumor tissues rather than the abdominal organs, owing to the abundant blood flow around the abdomen that facilitated the drug delivery. Therefore, the tumor tissues exhibited the greatest accumulations in Dir/C-NPs group, which was nearly 2.9-fold higher than the free Dir group, and the liver fluorescent intensity was 2.5-fold fold than the free Dir group.
Ex vivo tumor penetration
To estimate whether the ultrasound treatment could endow the redox/enzyme/ultrasound responsive self-destructive C-NPs a deep-penetrating ability, intact A549 solid tumors were dissected and incubated with C6/C-NPs with/without SDT treatment followed by frozen section. As depicted in Fig. 8D, ultrasound promoted the C6 penetration to deeper layers of A549 solid tumors by accelerating the blood flow in tumor vessels. For slices without SDT treatment, the C6 fluorescence was mainly observed at the surface of tumors with a narrow penetrating depth of 60.1 µm, while the depth of C6 in SDT-treated tumors reached to 231.3 µm from the outer layers to the inner part. Further, the penetrating behavior was quantitatively described by Image J software. Two lines were drawn from the surface of tumor slices to the inner layers, and the fluorescent intensity along with the lines was plotted at different distance from the outer layer of tumors (Fig. 8E). The signal attenuation trends of C6 in C-NPs were in line with the fluorescent result, suggesting the penetration promoting effect of ultrasound in tumor cells.
Anti-tumor efficiency
The in vivo anti-tumor of DTX/C-NPs by bilateral A549 tumor-bearing mice models was evaluated (Fig. 9A). The relative tumor volume (RTV) of bilateral tumor-bearing mice (‘Right (R)’ for in situ tumors and ‘Left (L)’ for distal tumors) was measured, and the body weight changes were also recorded (Fig. 9B to 9D). Conceivably, the tumor suppression effect was similar for ‘R’ and ‘L’ tumors, and the DTX/C-NPs with/without SDT groups showed decrescent RTV after 14 days compared with the control group (NS) with RTV of 4.40, 3.94, 1.62, 1.28, 0.74 and 0.77 for NS (R), NS (L), DTX/C-NPs (R), DTX/C-NPs (L), DTX/C-NPs + SDT (R) and DTX/C-NPs + SDT (L), respectively. Whereas, the C-NPs groups exhibited considerably lower RTV compared with Taxotere® and DTX + Rh groups especially for the L tumors in C-NPs group, which was probably contributed to the smaller size of distal tumors (Fig. 9E). Subsequently, the body weights of mice except for the groups of Taxotere® and DTX + Rh exhibited no significant change compared with NS group. The curves of Taxotere® and DTX + Rh showed that mice gained the minimum body weights at day 10 but regained the loss after the administration stopped. Therefore, the free drug possessed cumulative toxicity to mice, but could be metabolized quickly at the end of the administration.
Further, the expressions of COX-2 and uPA proteins in ‘L’ tumor tissues was evaluated via western blot assay with for observing the tumor metastasis-related indicators by preparing the homogenate (Fig. 9F). The expression of both COX-2 and uPA proteins was decreased in C-NP + SDT and DTX/C-NPs + SDT groups compared with NS group, manifesting the good anti-metastic effect of the SDT and chemo-sonodynamic combined treatment. Meanwhile, H&E staining assay was conducted and showed no obvious pathological damage of the main organs of (Fig. S9). Additionally, all ‘R’ and ‘L’ tumors were stained with H&E staining, and the slices images showed wonderful pro-apoptotic effect with a small amount of blue nucleus in DTX/C-NPs and DTX/C-NPs + SDT groups. Besides, C-NPs + SDT also presented reduced nucleus compared with NS group. However, the pro-apoptotic effect of SDT and combined therapy for ‘L’ tumors was less distinct than the ‘R’ results (Fig. 9G and 9H). TUNEL assay showed similar results both for ‘R’ and ‘L’ tumors (Fig. 9I and Fig. S10).
Figure 9 The summary of in vivo anti-tumor effect with bilateral A549 mice model. (A) Overall schedule of in vivo tumor inhibition study. RTV curves of ‘R’ (B) and ‘L’ (C) tumors and body weight curves (D). (E) Photographs of ‘R’ and ‘L’ tumors after the autopsy. (F) Western blot result of COX-2 and uPA proteins expression after treatment with different solutions at day 15. H&E stained images of ‘R’ (G) and ‘L’ (H) tumor tissues after the treatment with NS, C-NPs (SDT+), Taxotere, DTX + Rh, DTX/C-NPs (SDT + or SDT-). Scar bar, 100 µm. (I) TUNEL results of ‘R’ tumor from all bilateral tumor-bearing mice treated with NS, C-NPs (SDT+), Taxotere, DTX + Rh, DTX/C-NPs (SDT + or SDT-). Scar bar, 100 µm. **p < 0.01, ***p < 0.001.
The apoptotic cells were stained with yellow or pale brown color and signed by red arrows or curves, while the normal cells were presented as blue nuclei. According to the result, the NS showed negligible tumor apoptosis, while SDT induced powerful cellular apoptosis observed by the larger brown areas in the slices. The synergetic therapy of DTX/C-NPs + SDT was the most effective in cell killing as seen from the large number of brown vacuoles with several apoptotic nuclei among them, but DTX/C-NPs group showed feeblish efficacy. Moreover, the tumor tissues in DTX + Rh group exhibited more apoptosis than Taxotere® group owing to the little production of ROS by the Rh.
Serum composition detection
Considering the RTV and H&E staining results for ‘R’ and ‘L’ tumors, it was speculated that the immune system of mice might be activated during the course of administration after SDT treatment. Thus, the serum composition of IL-10 and IL-12 was measured by Elisa kit aiming at evaluating the generation of M2 and M1 subtypes of macrophages that acted as the pro/anti-inflammatory cytokines after different treatment [36]. Results suggested that the IL-10 content decreased after SDT treatment with C-NPs or DTX/C-NPs, however the variation was on the opposite side for the IL-12 factor (Fig. 10A). Therefore, SDT could elevate the expression of M1 macrophages while decrease the M2 type, manifesting the effective enhancement in the immune capacity of macrophages of DTX/C-NPs group with the help of ultrasound. In addition, the serum biochemical indicators of ALT, AST, CREA and BUN were detected (Fig. 10B to 10D), and the result implied no significant differences among different treating groups, indicating the good safety to heart, liver or kidney of DTX/C-NPs + SDT administration.
Immunocytochemical staining
Based on the disparate result of Fig. 5B (expression of MMP9 and VEGFA proteins), the immunofluorescence technique was applied to the ‘L’ tumor tissues with anti-CD31 marker to make clear that whether the angiogenesis in tumors was inhibited after SDT treatment (Fig. 10E). The endothelial cells in platelet, shown as red fluorescence in the slices, disappeared after C-NPs + SDT, DTX/C-NPs and DTX/C-NPs + SDT treatment than the NS group, manifesting the good vascular inhibition effect of DTX/C-NPs and SDT. Thus, the angiogenesis was indeed inhibited after DTX/C-NPs + SDT treatment but not through the VEGFA pathway. In addition, the M2 macrophages in tumor tissues were also stained with anti-CD206 marker and imaged. In accord with the histogram in IL-10 factor, lesser M2 macrophages was produced after SDT treatment compared with the other groups as the green fluorescence increased.