Preparation and characterization of CCM@MSNs-ISOIM
First, ISOIM was loaded into MSNs to form MSNs@ISOIM, after which MSNs@ISOIM were coated with CCM vesicles using ultrasound to prepare the CCM@MSNs-ISOIM nanocomposites (Figure 1). The transmission electron microscope (TEM) images showed that particle size uniformity and particle dispersion of MSNs were satisfactory (Figure 2A). In addition, TEM also demonstrated that the membrane wraps the outer surface of the nanoparticles and forms a "shell core" structure, which signifies that the MSNs were successfully coated by the cancer cell membrane (Figure 2B and 2C). Results from SDS-PAGE (Figure 2D) showed that almost all CCM proteins in the CCM@MSNss nanocomplex were retained, and the particle size analyzer (Figure 2E) illustrated that the particle size of MSNs was 57.6±0.35 nm, while that of CCM@MSNs was 134.5±0.96 nm, close to that of the cancer cell membrane (132.6±1.12 nm). The potential of MSNs was -31.4±0.61 mV, while that of CCM@MSNs was -26.2±0.73 mV, similar to that of the cancer cell membrane (-27.8±0.56 mV) (Figure 2F). UV/vis spectra (Figure 2G) depicted that the absorption peaks of CCM@MSNs-ISOIM were 321 nm, 202 nm and 230.5 nm, which were consistent with the absorption peaks of the CCM vesicles, MSNs and ISOIM, respectively, further confirming the CCM@MSNs-ISOIM’s successful construction.
Drug loading and release
MSNs were used as a carrier for drug loading, hence, the entrapment efficiency (EE) and loading efficiency (LE) of ISOIM loaded on the CCM@MSNs-ISOIM nanocomplex were verified to be 93.8±4.7% and 45.4±2.6%, respectively (Figure 3A). Based on mesoporous silica materials, pH-Responsive systems usually involve on/off capping or gating (by functional groups[31], polyelectrolytes[32] and ring-shaped compounds[33]) or host–guest interactions (electrostatic[34], covalent bonding[35] and coordination bonding[36]). Due to the complex procedures, it is often costly to prepare the pH responsive systems on a large scale. In order to improve the consequent adsorption and conjugation of molecules, it is usually necessary for the surface of mesoporous silica materials to be modified by introducing additional functional groups, which might put the further applications at risk for the pharmaceutical industry[37]. In this sense, it is of practical significance to develop a simple way to construct the pH-responsive drug delivery system using non-functionalized mesoporous silica materials[38]. Thus, in order to build up an efficient and economical drug delivery system, mesoporous silica nanoparticles (MSNs) are considered more appropriate to construct a simple drug delivery system through the direct host–guest interactions occurring between MSNs and drug molecules[39]. The in vitro ISOIM release characteristics of CCM@MSNs-ISOIM and MSNs-ISOIM were then evaluated (Figure 3B). At pH 7.4, the ISOIM release rates of CCM@MSNs-ISOIM and MSNs-ISOIM within 60 h were 24.1±3.1% and 27.0±3.4%, respectively, whereas at pH 5.0, the release rates of CCM@MSNs-ISOIM and MSNs-ISOIM were 93.3±3.6% and 95.2±2.9%, respectively. The above results show that the release rate of ISOIM at pH 5.0 is faster than that at pH 7.4. According to the in vitro drug loading and release experiments, MSNs were found to be efficient drug carriers. The acidic environment of the tumor[40] is conducive to the release of ISOIM from CCM@MSNs-ISOIM, and MSNs possess certain characteristics in low pH value sensitivity.
Biocompatibility studies of CCM@MSNs-ISOIM in vitro
The phagocytosis and hemolysis tests verified the biocompatibility of CCM@MSNs-ISOIM nanocomposites in vitro. CCM@MSNs and MSNs were labeled with RhB (red fluorescence), respectively, after which RAW264.7 macrophages were treated with CCM@MSNs-RhB and MSNs-RhB for 6 h, respectively. When macrophages are treated with MSNs-RhB, most macrophages demonstrated diffuse red fluorescence, indicating that macrophages strongly phagocytose MSNs (Figure 4A and 4B). In contrast, a weak degree of red fluorescence was observed by CCM@MSNs-RhB, indicating that CCM vesicles effectively inhibited the phagocytosis of macrophages. Accordingly, CCM vesicles were observed to enable the nanomaterials’ ability of immune escape, reducing the clearance rate. The in vitro toxicity of CCM@MSNs and MSNs were also evaluated through the detection of cell viability as well as the hemolysis rate of erythrocytes. Next, the cell viability of OCI-LY10 cells was determined by CCK-8. Figure 4C depicts the cell survival rate of OCI-LY10 cells incubated with a series of concentrations of MSNs or CCM@MSNs for 24 h, of which the survival rate of OCI-LY10 cells treated with 100 μg/mL CCM@MSNs was found to be as high as 90%. As illustrated in Figure 4D, no obvious hemolysis was evident in erythrocytes treated with CCM@MSNs and MSNs. Additionally, the hemolysis rate caused by CCM@MSNs was less than 1%, lower than that caused by MSNs. Therefore, the obtained results further indicate that CCM@MSNs possesses excellent biocompatibility.
Verification of targeting and antigen functionalization of cancer cell membranes in vitro
In order to determine the targeting of CCM vesicles for lymphoma cells, DSPE-FITC (green fluorescence) was used to label the CCM vesicles. The nucleus of OCI-LY10 was stained blue using Hoechst 33342. As shown in Figure 5A, DSPE-FITC-CCM vesicles gathered around the nucleus of OCI-LY10, indicating that CCM vesicles may adhere to OCI-LY10 cells with obvious active targeting abilities. In order to verify the existence of specific membrane antigens on CCM vesicles, cancer cell membrane protein markers and intracellular markers were analyzed via Western Blot. As shown in Figure 5B, specific cancer cell membrane marker pan-cadherin and E-cadherin were observed[24, 41]. However, as an indicator of cytoplasmic protein, cytoskeleton protein β-actin is mostly found in the bands of tumor cell lysate, where cancer cell membrane extract contents are less. Therefore, after hypotonic lysis and gradient centrifugation, the cytoplasmic components of lymphoma cells were basically removed, whereas the proteins on the lymphoma cell membrane remained on its surface.
Effect of CCM@MSNS-ISOIM on the survival rate of OCI-LY10 cells in vitro
According to the results of Figure 5C, the cell proliferation inhibition rate of OCI-LY10 cells treated with different concentrations of ISOIM for 24 and 48 h were higher than that of cells without ISOIM treatment, and the rate increased with a rise in drug dose, suggesting that the inhibitory effect of ISOIM on the proliferation of OCI-LY10 cells was dose-dependent. For times under 48 h, the cell proliferation inhibition rate gradually increased as time progressed, while the rate at 48 h was higher than that at 24 h (P<0.05). The IC50 score of OCI-LY10 cells at 24 h and 48 h were 87 μg/mL and 33 μg/mL, respectively. Therefore, the concentration of 33 ug/mL of free ISOIM was chosen for the follow-up experimental study. At present, the clinical dosage forms of traditional Chinese medicine mainly stay in the traditional dosage forms such as ointment, soup, pill and powder, while the anti-tumor active components contained in some traditional Chinese medicine show such drawbacks as poor water solubility, low bioavailability and low specificity distribution. Besides, most of them are administered orally, and the active, ineffective and even toxic components of the preparation are also introduced into the body[42]. With the drug encapsulated in the nano-drug delivery system, the active components of traditional Chinese medicine can penetrate the reticuloendothelial system, which not only improves the solubility of the drug, but also enhance the performance of the drug in tumor targeting through passive and active targeting. In the meantime, the tumor microenvironment can be taken advantage of to regulate the drug release performance. In this way, the drug can be released either synchronously or sequentially, thus enhancing the curative effect of the drug on the tumor, mitigating the side effects of the drug on the normal tissue, and reducing the toxic and side effects caused by clinical treatment[43]. As mentioned above, in order to address poor water solubility, short half-life, poor stability, low bioavailability and the toxicity of ISOIM, nano-preparation (MSNs@ISOIM and CCM@MSNs-ISOIM) was modified for comparison with free ISOIM to further demonstrate the advantages of nanotechnology and biomimetic nano-modification technology[44]. In order to verify the effects of ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM in the same experiment, the concentration of ISOIM in both MSNs@ISOIM and CCM@MSNs-ISOIM was set to 33 ug/mL. As depicted in Figure 5D, compared to the PBS group, the cell survival rates of the ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM groups were 68.83±4.72%, 55.80±6.44% and 35.85±5.36%, respectively. In the same experiment, the effects of ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM were compared, where the ISOIM concentration of MSNs@ISOIM and CCM@MSNs-ISOIM was found to be 33 μg/mL. The cell survival rate of the MSNs@ISOIM and CCM@MSNs-ISOIM groups were significantly lower than that of the ISOIM group. In short, CCM@MSNs-ISOIM was observed to possess the most significant effects of anti-lymphoma. Similarly, compared to the PBS group, both the ISOIM and CCM@MSNs-ISOIM groups inhibited colony formation of OCI-LY10 cells, and the inhibitory effect of the CCM@MSNs-ISOIM group on OCI-LY10 clone formation was found to be the most significant (Figure 5E).
Effects of CCM@MSNS-ISOIM on cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and apoptosis proteins
After OCI-LY10 cells were treated with PBS, ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM for 24 h, DCFH-DA was used as a fluorescence probe to detect the levels of intracellular ROS. The results of the flow cytometry in Figure 6A demonstrated that ROS level in the ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM groups were significantly higher than that in the PBS group, where CCM@MSNs-ISOIM was observed to be the most significant. Accordingly, CCM@MSNs-ISOIM was suggested to have the strongest effect on ROS production in OCI-LY10 cells. The results of the Annexin V-FITC/PI double staining flow cytometry showed that compared to PBS, the number of living cells decreased significantly, while the number of apoptosis cells increased significantly following ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM intervention for 48 h, from 5.76 ±0.46% and 10.70±0.48 to 21.48±1.27% (Figure 6B). Moreover, CCM@MSNs-ISOIM was confirmed to induce apoptosis in OCI-LY10 cells. After OCI-LY10 cells were treated with ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM for 48 h, the proportion of cells in the G2/M phase was 17.15±0.9%, 22.50±1.02% and 31.93±2.64%, respectively. Compared to the PBS group, the above results showed a gradually increasing trend in the G2/M phase, whereas the cells in the G0/G1 phase showed a relatively decreasing trend. Hence, CCM@MSNs-ISOIM was suggested to induce cell cycle arrest of OCI-LY10 cells in the G2/M phase (Figure 6C). Furthermore, the effects of ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM on MMP of OCI-LY10 cells by flow cytometry with JC-1 staining were evaluated. As shown in Figure 6D, the percentage of reduction to mitochondrial membrane potential reached 41.3±2.5% after treatment with CCM@MSNs-ISOIM nanocomplex, which was higher compared with ISOIM group (15.6 ±1.6%). To this effect, CCM@MSNs-ISOIM was inferred to damage the mitochondria of cells, leading to a decrease in MMP. Subsequently, the effects of CCM@MSNs-ISOIM on the level of apoptosis-related proteins were observed via Western Blotting analysis (WB). Compared to PBS, ISOIM and MSNs@ISOIM, CCM@MSNs-ISOIM significantly upregulated the expression of p53, caspase-9 and caspase-3 in OCI-LY10 cells (Figure 6E). According to the above results, after CCM@MSNs-ISOIM acts on OCI-LY10 cells in vitro, it was concluded that the induced ROS blocks the cell cycle and reduces MMP to promote the release of tumor suppressor gene p53 and caspase proteins, resulting in OCI-LY10 apoptosis.
Verification of CCM@MSNs-ISOIM targeting and its anti-tumor effects in vivo
In vitro experiments have confirmed that CCM@MSNs are able to target tumor cells and possess the ability of immune escape, therefore, they may accumulate at the tumor site. To test this hypothesis in vivo, Cy5 was used to label CCM@MSNs and MSNs, respectively, with Cy5-MSNs as the control group. Their biological distribution in vivo was then evaluated using near-infrared fluorescence imaging. As shown in Figure 7A, compared to Cy5-MSNs, the fluorescence intensity of the tumor site gradually increased following tail vein injection of CCM@MSNs-Cy5, indicating that CCM@MSNs accumulated more in the tumor than MSNs. Furthermore, 48 h after intravenous administration, the retention of CCM@MSNs in tumor tissue was found to be significantly higher than that of MSNs. In addition, bright red fluorescence was observed in the tumor tissue treated with CCM@MSNs-Cy5, which was more obvious than that in MSNs-Cy5 (Figure 7B), indicating that CCM@MSNs could efficiently deliver drugs. The above results demonstrate that CCM@MSNs nanocomposites have the active tumor targeting abilities as well as a high efficiency in drug delivery and drug release.
Afterward, an anti-tumor test in vivo was carried out on OCI-LY10 tumor-bearing nude mice using the CCM@MSNs-ISOIM nanocomplex. When the tumor tissues were treated with PBS, ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM respectively, compared to the free ISOIM, both MSNs@ISOIM and CCM@MSNs-ISOIM were observed to significantly inhibit tumor growth, of which CCM@MSNs-ISOIM had the most significant inhibitory effect (Figure 7C). Simultaneously H&E staining showed that the number of necrotic cells in the CCM@MSNs-ISOIM group was more than that in the other groups (Figure 7D), further suggesting that CCM@MSNs-ISOIM possesses better effects in anti-lymphoma than that of the free ISOIM.
CCM@MSNs-ISOIM can block the OCI-LY10 cell cycle and inhibit cell proliferation in vitro, hence, Ki-67 immunohistochemical staining was used to detect cell proliferation in vivo[45]. As shown in Figure 7E, the number of Ki-67 positive cells (brown) in the CCM@MSNs-ISOIM group was found to be significantly less than that in the other groups, suggesting that CCM@MSNs-ISOIM most significantly inhibited the proliferation of lymphoma cells. In order to verify the pro-apoptotic effect of CCM@MSNs-ISOIM in vivo, TUNEL staining was utilized to detect apoptosis[46]. As shown in Figure 7F, nuclear staining of tumor cells in CCM@MSNs-ISOIM group was found to be positive (brown), which was significantly higher than that in the ISOIM group. The results of TUNEL were consistent with the apoptosis of lymphoma cells induced by CCM@MSNs-ISOIM in vitro.
Anti-Lymphoma Mechanism of CCM@MSNs-ISOIM
Apoptosis is one of the main ways in inhibiting the growth of tumor cells, and mitochondria are the most important organelles in the regulation of this process[47]. In addition to supplying energy to cells, mitochondria produce ROS, an apoptosis-inducing signal molecule[48]. ROS can directly trigger the opening of mitochondrial permeability transition pores and decrease the mitochondrial membrane potential, resulting in the activation of the mitochondrial Caspase-dependent apoptosis pathway[49, 50]. CCM@MSNs-ISOIM has a specific and powerful anti-tumor outcome in vitro, producing ROS to block the cell cycle while reducing MMP to activate apoptosis-related proteins, which promotes the mitochondrial apoptosis pathway against lymphoma. In order to verify whether CCM@MSNs-ISOIM also inhibits tumors via mitochondrial apoptosis pathway in vivo, the expression levels of ROS, MMP, Caspase-9 and Caspase-3 were detected. As shown in Figure 8A, after the tumor was treated with PBS, ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM for 21 days, the red fluorescence signal in the tumor tissue section of the CCM@MSNs-ISOIM group was most evident, indicating that CCM@MSNs-ISOIM produced more ROS than ISOIM alone. Then, a small amount of green fluorescence was observed in the tumor tissue sections of the ISOIM group, whereas diffuse green fluorescence was observed in the tumor tissue sections of the CCM@MSNs-ISOIM group, suggesting that, compared to the other groups, CCM@MSNs-ISOIM contributed to the most significant decrease in MMP (Figure 8B). By conducting an immunofluorescence assay to detect the expression of apoptosis protein Caspase-9 and Caspase-3, red (Caspase-9) and green (Caspase-3) fluorescence of the tumor tissue slices in the CCM@MSNs-ISOIM group depicted a strong level of fluorescence, while that of Caspase-9 and Caspase-3 in the other groups were significantly weaker, indicating that these two genes were highly expressed in tumor tissues treated with CCM@MSNs-ISOIM (Figure 8C). P53 protein acts as the guardian of the genome while playing an important role in regulating cell proliferation[51]. Cellular stress like DNA damage and carcinogenic signals can activate the p53 tumor suppressor pathway and coordinate the transcriptional response of hundreds of genes[52]. The study further found that p53 activation induces DNA repair or apoptosis by initiating multiple pathways[53]. In view of the important role of p53 protein activation in cell cycle regulation, the effect of CCM@MSNs-ISOIM on p53 protein activation in NHL was discussed. Figure 8D showed that the fluorescence intensity (pink) of p53 in the CCM@MSNS-ISOIM group was more significant than that in the other groups, indicating that CCM@MSNS-ISOIM enhanced the expression of the p53 protein. CCM@MSNs-ISOIM may also induce DNA damage by inducing G2/M phase arrest, promoting the activation of p53 protein, which further leads to apoptosis of lymphoma cells. The corresponding results suggest that CCM@MSNs-ISOIM induces apoptosis of lymphoma through the G2/M/p53 and mitochondrial apoptosis pathways.
Study on the biosafety of CCM@MSNs-ISOIM in vivo
To verify whether CCM@MSNs-ISOIM has potential toxicity in vivo, the toxicity of PBS, ISOIM, MSNs@ISOIM and CCM@MSNs-ISOIM were evaluated via tail vein injection in nude mice. A histological analysis was then carried out and compared to the PBS group, which demonstrated no significant pathological damage in the major organs of the nude mice treated with CCM@MSNs-ISOIM for 3 weeks (Figure 9A). In addition, no significant abnormal changes in the body weight of the nude mice were present in the above four groups within 3 weeks (Figure 10A). Furthermore, no abnormal changes were present in WBC, HGB, PLT, ALT, AST, BUN, CRE, CK and Myo in each group (Figure 10B and 10C), signifying that CCM@MSNs-ISOIM is non-toxic and possesses good biocompatibility.