| OBJECTIVESo far, chemotherapy is still one of the main methods of systemic therapy for malignant lymphoma. However, chemotherapy is often accompanied by severe side effects, which limits its effective application in clinical practice. To reduce severe side effects, improve the active targeting of antitumor drugs, and enhance antitumor efficacy, we constructed a controlled-release and targeted drug delivery system based on mesoporous silica nanoparticles.Rituximab-conjugated and doxorubicin(DOX) hydrochloride-loaded mesoporous silica nanoparticles (RDMSNs) were obtained. The drug delivery system can both target specifically to lymphoma B cells and release drug for the treatment of B cell lymphoma under intracellular acidic environment. In this paper, the targeted drug delivery system was used to treat B cell lymphoma, to evaluate its antitumor effect and to explore its antitumor mechanism.METHODS1. The construction and characterization of RDMSNs targeted drug delivery system:Firstly, mesoporous silica nanoparticles modified with carboxyl groups were synthesized by copolymerization method, and used as carriers for constructing an intracellular pH responsive drug delivery system. Based on this, Biotin-PEG2000-Amine was modified to the nucleus surface to optimize the nanoparticles. Next,rituximab,which could specifically target to the tumor cell membrane with high expression CD20 receptor, was conjugated to the surface of nanoparticles to prepare the RDMSNs with a target ability to lymphoma B cells using biotin-avidin bridging method. The prepared nanoparticles were characterized by scanning electron microscopy, transmission electron microscopy, fourier transform infrared spectroscopy, laser particle size analyzer and UV spectrophotometer, respectively. The nanoparticles encapsulation efficiency, drug loading efficiency and the binding efficiency of antibody attachment to the nanoparticles were calculated. The drug release characteristics of nanoparticles were evaluated using dialysis experiments under different pH value conditions.2. Evaluation of cell targeting and in vitro cytotoxicity of RDMSNs: The targeting function of RDMSNs were evaluated using CD20+ Raji cells and CD20 negative Jurkat cells,and the fluorescence intensity of the cells were observed by flow cytometry and confocal laser scanning microscopy, and the number of intracellular nanoparticles was observed by transmission electron microscopy. To verify the cellular targeting and in vitro cytotoxicity of RDMSNs, the cytotoxic effect of drug loaded nanoparticles was evaluated in CD20 positive Raji and Daudi and CD20 negative Jurkat cells using CCK-8 kit. The nuclear morphological changes of cell apoptosis were observed under confocal laser microscopy and the cell apoptosis was also detected by flow cytometry.3. Study of RDMSNs on Raji cell lymphoma and in vivo fluorescence imaging: An in vivo murine Raji lymphoma model was established to explore the antitumor efficacy of nanoparticles. When lymphoma volume reached approximately 100 mm3, Raji lymphoma bearing mice were randomly divided into 4 groups (5 mice per group), respectively : Saline group, Free DOX group, DMSNs group, and RDMSNs group. Each Raji lymphoma bearing mouse was treated with DOX related formulations (the dose was 2.0 mg / kg) once every 4 days for a total four times to detect the antitumor effect. The lymphoma volume were examined to evaluate the tumor inhibition rate. After 16 days of treatment, the main organs including the heart, liver, spleen, lungs and kidneys were acquired for hematoxylin-eosin staining to detect the toxicity in vivo. Additionally, the cell apoptosis was detected in lymphoma tissue using a TUNEL apoptosis detection kit, the expression of apoptosis-related proteins like Bax, Caspase-3, Bcl-2 and nuclear Ki67 antigen was detected using immuno-histochemical method. In addition, near infrared imaging in nude mice was used to evaluate the targeting properties of nanoparticles.RESULTS1. MSN-COOH nanoparticles with well-defined structure were successfully prepared which were used as carriers to prepare RDMSNs. Scanning electron microscopy and transmission electron microscopy both showed MSN-COOH nanoparticles had a uniform and regular shape. FTIR spectroscopy confirmed that the carboxyl groups were successfully connected to MSN, the mean diameter and zeta potential of RDMSNs were 56.3±11.2 nm and -31.5 ± 5.2 mv,respectively. The encapsulation efficiency was approximately(45.2±6.2)%,and the loading efficiency was approximately (23.5±4.7)%. Rituximab binding efficiency was approximately (66.2 ± 4.1)%. In the drug release experiment, the results demonstrated that the DOX released from RDMSNs was significantly higher at a pH of 5.0 compared with that at a pH of 7.4 (P < 0.05 ),suggesting that RDMSNs possessed pH-responsive drug release property.2. In Raji and Jurkat cell lines, after the cells were treated with RDMSNs, the results showed that the fluorescence intensity in Raji cells was approximately 3 times that of Jurkat cells by flow cytometry (P<0.01 ) , and that of Raji cells was significantly higher than that of Jurkat cells under confocal laser scanning microscopy. Moreover, the similar results were also obtained by transmission electron microscopy. In Raji, Daudi and Jurkat cell lines, the cytotoxic effect of RDMSNs in Raji and Daudi cell lines was higher than those of DMSNs and Free DOX (P< 0.05 ) . Furthermore, the results exhibited a DOX dose-dependent cytotoxicity, but there was no significant difference of cytotoxic effect between DMSNs group and RDMSNs group in Jurkat cells. In the apoptosis experiment, the morphological changes of apoptosis in RDMSNs group were more significant than those of other groups under confocal laser scanning microscopy. The apoptosis rates of Raji cells treated with PBS,MSNs,Free DOX,DMSNs and RDMSNs for 24h was (3.03 ± 0.36)%,(3.94 ±0.54)%, (18.22 ±1.15)%,(10.09 ± 1.10)%,and (23.29±1.37)%, respectively. The apoptosis rates in RDMSNs group were significantly higher than those of other groups (P < 0.05).Moreover, a concentration-dependent apoptosis efficiency caused by the RDMSNs was also detected.3. In the in vivo efficacy evaluation experiment, The tumor volume of mice in Saline group, Free DOX group and DMSNs group were 623.5± 156.9mm3, 481.2±55.2mm3and 335.6±57.3mm3, respectively,while the tumor volume of mice in the RDMSNs group was 98.0±51.8 mm3, and the difference was statistically significant compared with other groups (P< 0.05). The weight of mice in Saline group, RDMSNs group and DMSNs group were 25.22±0.9g?22.4±0.5g and 21.6±0.5g, respectively,while the weight of mice in the Free DOX group was 18.1 ±0.6g,and the difference was statistically significant compared with other groups (P < 0.05). Tumor inhibition rates of Free DOX, DMSNs and RDMSNs group were (22.79±4.37) % , (46.22±6.03) % and (84.28±5.92) % , respectively. Compared with other two treatment groups, the tumor inhibition rate of RDMSNs group was the highest (P <0.01). All these data suggested that RDMSNs had stronger antitumor effect, less side effects,and targeted antitumor performance. Hematoxylin-eosin staining of heart tissue in Free DOX group showed the myocardial fibers disorder, while H&E staining indicated there were no significant pathological lesions or damages in the major organs including heart, liver, spleen,lung and kidney in other groups. These results showed that the drug loaded nanoparticles had a good biocompatibility in vivo. In TUNEL staining experiment, the RDMSNs group indicated the most significant green fluorescence and the most apoptotic cells compared with other groups. Immunohistochemical analysis showed that the protein expression of Bax and caspase-3 in DMSNs and RDMSNs group was increased significantly compared with that in the Saline and Free DOX group, while that of Bcl-2 protein and Ki67 antigen was significantly reduced, interestingly, the protein expression of Bax and caspase-3 in group RDMSNs was the strongest compared with other groups, while the expression of Bcl-2 protein and Ki67 antigen was the weakest compared with other groups. These results indicated that RDMSNs possessed strong induction of apoptosis and anti-proliferative properties. In the near infrared imaging experiment, the fluorescence signal in Cy5.5-RDMSNs group specifically was accumulated at the tumor site after 24h and then the fluorescence signal gradually decreases. The similar imaging results were obtained in Cy5.5-DMSNs group, while the fluorescence signal of tumor site in Cy5.5-labeled DMSNs group after 6h was weaker than that of tumor site in Cy5.5-RDMSNs group using semi-quantitative fluorescence signal analysis (P < 0.05).CONCLUSIONS1. The configuration of RDMSNs was uniform and regular, having high drug loading.Due to the surface modification of rituximab and PEG, RDMSNs had pH responsive drug release performance.2. RDMSNs in vitro can specifically be internalized into lymphoma B cells, which may be completed via receptor-mediated endocytosis pathway. Intracellular endosomes or lysosomes formed can promote the drug release and increase the drug accumulation within cells owing to its acidic environment, which could play an important role in enhancing cytotoxic effect and inducing cell apoptosis.3. RDMSNs have the active targeting specificity to lymphoma B cells and enhanced antitumor efficacy, low toxicity in vivo, improving the induction of cell apoptosis.Controlled-release drug delivery system is likely to become a carrier of chemotherapy drugs,enhancing drug targetbility to tumor cells, improving the efficacy of drug, and minimizing the toxicity of drug, which may provide a new platform for the targeting therapy of B cell lymphoma. |
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