Effects of chemotherapeutic drug-curcumin nanoparticle conjugated with two peptides against flt3 protein on induction of leukemic stem cell death

Abstract

Leukemic stem cells (LSCs) are an important cause of relapse in acute myeloblastic leukemia (AML) patient. Anthracyclines, such as doxorubicin (Dox) and idarubicin (Ida), are the standard chemotherapy for AML treatment, and found less effective to destroy LSC, and might generate adverse effects in high dose treatment. Curcumin (Cur) is the main natural polyphenolic compound found in the rhizome turmeric. It is known having anti-tumor, chemo-sensitizing, and chemoprotective activity. Therefore, to enhance efficacy and reduce toxicity of drug, the combination treatment of anthracyclines and Cur were evaluated on AML leukemic stem cells. The cytotoxicity of single and co-treatment of Dox, Ida, and Cur were investigated in AML leukemic stem cells (KG-1a and KG-1) and AML leukemic cells (EoL-1 and U937). The cytotoxic study of single drugs showed that Ida, Dox, and Cur exhibited higher toxicity in EoL-1 and U937 cells than KG-1a and KG-1 cells, resulting from the drug resistant properties of LSCs. Thus, the cytotoxic effects of co-treatments of Dox or Ida at concentration of IC10-IC80 values and each concentration of Cur at IC20, IC30, IC40, and IC50 were assessed. All conditions of Dox-Cur and Ida-Cur were observed to have a tendency to increase cytotoxicity in cell models according to the increase of Cur dose, compared to single treatment. However, only Dox-Cur showed additive effects in several conditions of co-treatment. Thus, this co-treatment of Dox-Cur was selected to study effects on FLT3 protein expression in FLT3 expressing cell, such as KG-1a, KG-1, and EoL-1 cells. The results showed all conditions of Dox-Cur could lower FLT3 protein expression level and lived cell number in all cells. Cur was expected as a main compound that affected FLT3 protein expression. In addition, co-treatment of Dox-Cur could induce G2/M cell cycle arrest and apoptosis in KG-1a and EoL-1 cells, that represent AML stem cells and leukemic cells, respectively, when compared to a single treatment and a control. Moreover, Dox-micelle (DM), Cur-micelle (CM), and Dox-Cur-micelle (DCM) were developed to improve drug uptake efficacy both compounds in leukemic cells. Besides, the drug-micelles were conjugated with FLT3 specific peptides (CKR and EVQ) to increase drugs deliver to leukemic cells through FLT3 receptor. DM, CM, and DCM were formulated by using a film-hydration technique together with a pH-induced self-assembly method. The optimal drug-to-polymer weight ratios for DM, CM, and DM formulations were 1:200, 1:40, and 1:40, respectively. The weight ratio of Dox and Cur in DCM was 1:9, which was derived from the lowest concentration of Dox and Cur that exhibited higher cytotoxicity than a single Dox treatment. For the examination of physicochemical properties, DM, CM, and DCM exhibited a particle size in the range of 20-25 nm with a narrow size distribution, neutral charge, and a high %EE (more than 80%), and -80°C was the effective micelle storage temperature. In addition, each micelle exhibited colloidal stability and could prolong drug release. Thus, these DM, CM, and DCM formulations were suitable for peptide conjugation. To construct a micelle conjugated with FLT3 peptides, P407 was converted to cargo azide (CA) and linked to FLT3 peptides to produce CA-CKR and CA-EVQ. CA-CKR and CA-EVQ were combined to provide micelle with two peptides (CA-CKR+CA-EVQ). Then, micelle was developed. DM, CM, and DCM coupled with peptide CKR (C), EVQ (E), and CKR+EVQ (C+E) exhibited a higher particle size and a slightly lower drug content compared to micelles without peptide, which may be attributable to the incorporation of the peptides. The assessment of cytotoxic effect revealed that DM-C+DM-E exhibited the highest cytotoxicity in KG-1a cells. DCM conjugated with or without peptides had no effects on KG-1a and EoL-1 cells due to an uncontrolled drug concentration ratio. Therefore, the cytotoxicity of co-treatment of several DM and CM was further investigated in KG-1a cells. The results showed that DM-C+CM-C showed the highest cytotoxicity. Moreover, DM-C+DM-E and DM-C+CM-C had the highest efficacy to induce apoptosis in KG-1a cells. The used of co-treatment of DM and CM could decrease concentration of Dox use in leukemic cell treatment. However, micelles conjugated with CKR or both CKR and EVQ were found no statistically significant difference in dose use for cytotoxic and apoptosis induction studies. These results suggested that the combination of micelle and FLT3-specific peptides could enhance Dox and Dox-Cur uptake by AML leukemic stem cells. The combination of the two peptides might enhance drug uptake activity and reduce the disadvantages of the individual peptides when stored for an extended period of time.