| OBJECTIVEChemotherapy offers a systemic cancer treatment. However it is limited in clinical administration due to its serious side effects. Nanoparticles drug delivery systems(DDS) can sustainedly release anticancer drug at the specific site and reduce the incidence of toxicity on normal tissues. To evaluate the benefit of a novel chemotherapeutic DDS and its underlying mechanisms, daunorubicin(DNR) was loaded into PLGA-PLL-PEG-Tf nanoparticles (NPs) to construct DNR-PLGA-PLL-PEG-Tf-NPs (DNR loaded NPs) as a DDS.METHODSThe physical properties of DNR-PLGA-PLL-PEG-Tf were determined by transmission electron microscope(TEM), scan electron microscope(SEM), laser particle size analyzer and UV spectrophotometer, respectively. In vitro, the intracellular concentration of DNR in leukemia K562 cells was detected by flow cytometry (FCM) and observed under fluorescent microscopy. In vivo, the effect of drugs on the growth of tumors in K562 xenografts was observed and the relevant toxicity of therapeutic drugs to organs was investigated. Moreover, cell apoptosis in the excised xenografts was measured by TUNEL assay. Apoptosis related protein expression levels of Bcl-2, Bax, Caspase 9, Caspase 3 and cleaved-PARP(c-PARP) were determined by western blotting. Caspase 3 was determined by immunohistochemistry. Leukemia primary cell apoptosis and the intracellular concentration of DNR were determined by FCM.RESULTS1. DNR loaded NPs had a spherical shape and dispersed uniformLy under TEM and SEM, the mean size and ZP of DNR-PLGA-PLL-PEG-Tf NPs (DNR loaded NPs) were 176.4±11.0 nm and-19.24 ± 0.12 mV, respectively. The encapsulation efficiency was 75.12% ± 1.68% and the drug loading was 5.32% ± 0.15%. These results demonstrate that DNR loaded NPs is relatively homogenous in size and can be stored stably.2. Under fluorescent microscope, fluorescence was observed in K562 cells treated with DNR and DNR-PLGA-PLL-PEG-Tf. The RFI (FI treated group/FI control group) of intracellular DNR in K562 cells was higher in DNR loaded NPs group than that in DNR group(14.26 ± 0.39 vs 8.14 ± 0.29), and there was a significant difference between them (p<0.05). Otherwise, there was no significant difference between PLGA-PLL-PEG-Tf-NPs and control group (p>0.05). These results suggest that DNR modified by PLGA-PLL-PEG-Tf-NPs can be targeted successfully to leukemia cells. 3. In vivo, the mean tumor volume was 1,099 ± 238 mm3 for saline water,1,034 ± 178 mm3 for PLGA-PLL-PEG-Tf-NPs,536 ± 137 mm3 for DNR, and 237 ± 85 mm3 for DNR loaded NPs, and the inhibition of tumor growth in either DNR or DNR loaded NPs group was significantly higher than that in PLGA-PLL-PEG-Tf-NPs and control groups (p＜0.05). Interestingly, the mean tumor volume in DNR loaded NPs group was smaller than that in DNR group (p<0.05), while there was no significant difference between PLGA-PLL-PEG-Tf-NPs and control groups (p>0.05). These results were further demonstrated by the weight of the tumors. Thus, we infer that the DNR loaded NPs have a greater antitumor efficacy than DNR (p＜0.05). The apoptotic rate in DNR loaded NPs group increased significantly compared with that in DNR group (p<0.05). However, there was no significant difference between PLGA-PLL-PEG-Tf-NPs and control groups (p>0.05), indicating that DNR loaded NPs show more effective drug treatment to achieve cell apoptosis than DNR (p<0.05).To explore the possible signaling pathways through which DNR loaded NPs induced more anticancer activity, we examined the changes in the expression levels of apoptosis related proteins by western blotting and immunohistochemistry. In our study, the level of apoptosis regulating protein Bcl-2 in both DNR group and DNR loaded NPs group were downregulated while that of Bax was upregulated compared with the control group (p＜0.05). In addition, the expression of Bcl-2 and Bax proteins in DNR loaded NPs group was more obviously regulated than that in DNR (p＜0.05).The expression of Caspase 9 was upregulated in treatment groups compared with that in the control group (p<0.05), and the increase was much more dramatically in DNR loaded NPs group than that in DNR group(p<0.05). Similar results of Caspase 3 and c-PARP were detected. Notably, the protein expression of PLGA-PLL-PEG-Tf-NPs group was not obviously changed when compared with the control group (p>0.05). All the mice were alive and they were weighed during the whole experiment period. It is noteworthy that no significant differences in body weight and no abnormal finding in any group. There were no apparent histopathologic changes in the tissues including lung, heart, liver, kidney and spleen. These results indicate that DNR loaded NPs are safe and have no obviously toxicity to the main organs of mice.4. Apoptosis in leukemia primary cells treated with DNR and DNR loaded NPs were detected by FCM, and there was no significant difference between them. The RFI of intracellular DNR determined by FCM was higher in DNR loaded NPs group than that in DNR group, and there was a significant difference between them (p<0.05).CONCLUSIONS1. In the present study, spherical DNR loaded NPs with diameters of 176.4±11.0 nm is produced with a remarkably high DNR encapsulation efficiency and loading, which is suitable for drug delivery. The delivery of DNR using NPs amplified the DNR antitumor that possibly elicited systemic targeting in leukemia.2. In vitro, DNR loaded NPs showed higher DNR accumulation than the DNR alone. In vivo, the tumor volume of the mice injected with DNR loaded NPs was smaller than that of the mice treated with DNR. We infer that DNR loaded NPs can be directly delivered into tumor cells by receptor-mediated endocytosis and drug sustained release from nanoparticles with the degradation of nanoparticles skeleton. Thereby, DNR loaded NPs may render longer half-life of DNR in blood and DNR concentration must be increased in the tumor tissue.3.The level of Caspase 9, Caspase 3 and its substrates c-PARP proteins was remarkably elevated in DNR loaded NPs group compared with other groups. In the present study, we observed the expression of pro-apoptotic and anti-apoptotic members of the Bcl-2 family.Our present results showed that the level of Bcl-2 protein was downregulated in DNR loaded NPs group compared with DNR group, whereas that of Bax was upregulated, and the ratio of Bax to Bcl-2 was increased. Taken together, these data indicate that DNR loaded NPs can activate the intrinsic signaling pathway to trigger leukemia cell apoptosis.4. The novel NPs DDS enhance the activity of traditional anticancer drug DNR against leukemia cells through activating the intrinsic apoptosis by both active and passive targeting pathway. This would ensure the improved efficacy and minimize toxicity, which may be a useful clinical tool. |
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