| OBJECTIVE To develop natural platelets as a smart drug delivery system to load doxorubicin (DOX) for the treatment of lymphoma, the characterization was investigated. The possible morphological and functional changes of platelets induced by DOX were estimated. The efficacy of DOX-loaded platelets on Raji cells and tumor-bearing mice were studied, and the cardiotoxicity was also tested on myocardial cells and tumor-bearing mice.METHODS (1) Washed platelets were gained after centrifuging and washing blood, afterwards, thry were mixed with different contentrations of DOX, and by passing through a Sepharose 2B column, DOX-platelet was obtained; (2) High-performance liquid chromatography and electronic scale were utilized to evaluate the drug loading (DL) and encapsulation efficiency (EE). Fluorescence microscopy was performed to determine whether the drugs were loaded in platelets and the possible morphological changes induced by DOX on platelet were investigated by scanning electron microscopy (SEM). The expression of some membrane proteins of platelets (CD41, CD61 and CD47) both in platelet and DOX-platelet were detected by western blotting. Platelet aggregation was estimated by a spectrophotometric method. Dynamic dialysis methods were used to investigate the release behavior of DOX from the platelets in vitro. (3) The Cell counting kit-8 assay was used to evaluate cell viability. (4) Flow cytometry (FCM) was used to quantitatively detect cell apoptosis; (5) Morphological changes of Raji cells were observed under a confocal microscope after DAPI staining. (6) Cellular uptake was quantitatively measured by FCM. (7) The level of mRNAs and protein expression of Bad, Bcl-xl, Caspase9 and P53 were measured by Reverse transcription-polymerase chain reaction (RT-PCR) and by Western Blot, respectively. (8) When tumor models were established successfully, they were divided into 4 groups with different treatments, and the weight of mice and volume of tumors were measured every two days. (9)The major organs of mice were isolated and stained with H and E tainting for histopathological examination. (10)The expression levels of mRNAs and proteins of Bad, Bcl-xl, Caspase-9 and P53 in vivo were evaluated by RT-PCRand western blot analysis.RESULTS(1) Characterization of DOX-platelet?. DOX could be loaded into platelets with the DL of 46.3% and EE of 86.6%;?. No significant morphological changes of platelets were induced by DOX, the aggregation behavior of platelet and DOX-platelet were reality. There were no remarkable changes of the expression of CD41, CD47 and CD61 in platelet and DOX-platelet.?. In the conditon of pH 5.5, approximately 84.4% of DOX was released from DOX-platelet within 36 h, however, the release was less than 40% at pH 7.4 and 8.4..(2) Growth inhibition on Raji cells and myocardial cells?. The growth inhibition efficacy of DOX on Raji cells was in a time and dose-dependent manner, and the IC50 of 24h was calculated as 0.243ug/ml.?. The incubation of Raji cells and myocardial cells with platelets caused no significant reduction in viability compared with controls.?. After incubation for 24,48, and 72 h, the corresponding inhibition rates by DOX were (52.06±3.5)%, (61.90±4.0)% and (72.30±4.1)%, compared to (65.80±3.0)%, (85.23±3.7)% and (90.60±3.4)%, respectively, for the DOX-loaded platelets. Both DOX and DOX-platelet showed a significant difference with controls. A significant difference was also observed between the DOX and DOX-platelet groups. (At 24h and 48h, P<0.05; at 72 h, P<0.01)?. After incubation for 24,48, and 72 h, DOX-treated group showed more inhibitions on myocardial cells when compare with DOX-platelet. (P<0.01)(3) Raji cell apoptosis?. After incubation for 24 h, the total apoptosis rates of controls, platelet, DOX and DOX-platelet were (5.9±0.52)%, (14.6±1.21)%, (50.5±5.01)% and (73.1±8.37)%, respectively, DOX and DOX-platelte showed a significant difference compared with controls. A significant difference was also observed between the DOX and DOX-platelet groups (P<0.01).?. Chromatin condensation, nucleolus pyknosis and nuclear fragmentation were observed on Raji cells when DOX introduced, and the morphological changes were more drastic when DOX-loaded platelets.(4) Cellular uptakeThe intracellular DOX concentration significantly increased in the DOX-loaded platelet group compared with the unencapsulated DOX group.(5) RT-PCR and western blot assay of Raji cellsThe mRNA expression of Bad, caspase-9, and p53 was upregulated to some extent in the DOX group compared with that in the control and to a greater extent in the DOX-loaded platelet group (P< 0.05). By contrast, Bcl-xl transcription was downregulated in the DOX group compared with the control, and its expression level was further downregulated when DOX was carried by platelets. Similar changes of all apoptosis-related genes were also observed by western blot analysis.(6) Changes of body weight and tumor size of mice after treatments?. A smaller tumor size was observed in DOX and DOX-platelet when compared with controls, it was more dramatic in DOX-platelet.?. Only DOX group showed asignificant weight loss in comparison with controls.(7) Histopathological studyTypical DOX-induced myocardial injury to mouse hearts only could be observed in the DOX group. No significant histopathological abnormalities were observed on any other organs in any group.(8) RT-PCR and western blot assay of tumor bodiesThe mRNA expressions of Bad, Caspase-9 and P53 were upregulated to some extent in DOX compared with controls, and it was dominant especially in the group of DOX-platelet. The transcription of Bcl-xl was downregulated in DOX group when compared with controls, and the expression level was further downregulated when DOX was carried by platelet. The same variation tendency was also observed in western blot assay.CONCLUSION (1) No significant morphological and functional changes of platelets were induced by DOX. (2) DOX-platelets could induce intracellular drug accumulation, reduce adverse effects and enhance therapeutic efficacy, as verified by in vitro and in vivo experiments. (3) DOX-loaded platelets improved the anti-tumor activity of DOX through regulating the expression of apoptosis-related genes such as Bad, Caspase9, Bcl-xl and P53. |
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