Idarubicin-loaded methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide) nanoparticles for enhancing cellular uptake and promoting antileukemia activity

Figures & data

Figure 1 (A) Synthesis route of mPEG-PLGA diblock copolymer and schematic diagram of preparation of IDA/mPEG-PLGA NPs. (B) ¹H NMR characterization of mPEG-PLGA diblock copolymers. (C) FTIR spectra of three kinds of mPEG-PLGA diblock copolymers.

Abbreviations: GA, glycolide; IDA, idarubicin; LLA, l-lactide; mPEG, methoxy poly(ethylene glycol); mPEG-PLGA, mPEG-b-poly(LLA-co-GA); NPs, nanoparticles.

Table 1 Size, ZP distribution, DL, and EE of NPs and/or IDA/mPEG-PLGA NPs

Sample ID	Size (nm)	PDI	ZP (mV)	DL (%)	EE (%)
mPEG-PLGA1 NPs	72.4±4.6	0.118±0.004	−33.8±2.1
IDA/mPEG-PLGA1 NPs	81.2±3.4	0.107±0.005	−34±3.6	8.59±0.96	93.95±3.2
mPEG-PLGA2 NPs	90.65±2.3	0.119±0.012	−30.1±2.6
IDA/mPEG-PLGA2 NPs	98.24±3.5	0.124±0.013	−31.6±2.8	8.31±0.68	91.72±2.6
mPEG-PLGA3 NPs	111.8±1.9	0.123±0.007	−32.5±2.4
IDA/mPEG-PLGA3 NPs	123.5±4.1	0.131±0.013	−32±3.5	8.28±0.75	91.09±3.4

Note: Size indicates NPs’ diameter, n=3.

Abbreviations: DL, drug loading; EE, encapsulation efficiency; IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles; PDI, polydispersity index; ZP, zeta potential.

Figure 2 TEM images of mPEG-PLGA₁ NPs (A) and IDA/mPEG-PLGA NPs (B).

Abbreviations: IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles; TEM, transmission electron microscopy.

Figure 3 Standard curve of IDA and cumulative release of IDA/mPEG-PLGA NPs.

Notes: (A) Standard curve of IDA; images of IDA and IDA/mPEG-PLGA NPs after centrifugation separation. (B) Cumulative release of IDA/mPEG-PLGA NPs. Abbreviations: IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles.

Table 2 IDA release in complete medium and PBS

	Day 2	Day 4	Day 7	Day 10
IDA release (%) (in complete medium)	22.1±3.1	35.0±1.2	46.2±0.6	55.12±2.4
IDA release (%) (in PBS)	20.1±0.6	37.1±0.9	48.9±0.2	53.1±0.8

Abbreviation: IDA, idarubicin.

Figure 4 Cytotoxicity of mPEG-PLGA₁ NPs, IDA/mPEG-PLGA NPs, and IDA in L929 cells and normal human CD34⁺ HSPCs.

Notes: (A and B) The cell viability was measured in murine L929 cells and normal human CD34⁺ HSPCs treated with different concentrations of mPEG-PLGA₁ NPs, IDA, and IDA/mPEG-PLGA₁ NPs for 24 hours (**P<0.01 vs control group; NS, nonsignificant between control group and IDA/mPEG-PLGA NPs).

Abbreviations: HSPCs, hematological stem and progenitor cells; IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles.

Figure 5 Pharmacokinetics and biodistribution analysis by ex vivo imaging system.

Notes: (A) Six-week-old wild-type C57BL/6J mice were intraperitoneally injected with IDA/mPEG-PLGA NPs. Liver, spleen, heart, lung, kidney, and intestine were dissected out after administration for 6 hours. The 540_Ex/620_Em was used to analyze DsRed fluorescence by IDA and captured by ex vivo imaging system. (B) Six-week-old wild-type C57BL/6J mice were intraperitoneally injected with IDA/mPEG-PLGA NPs. Bone marrow was dissected out after administration for 6 hours for subsequent ex vivo imaging analysis. (C) Ex vivo imaging analysis for liver and spleen, which were dissected out after mice were injected with IDA (3 mg/kg) or IDA/mPEG-PLGA NPs (3 mg/kg) for 2, 24, 48, and 96 hours.

Abbreviations: IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles.

Figure 6 mPEG-PLGA enhances the cellular uptake of IDA.

Notes: PE fluorescence by IDA was measured by flow cytometry in HL-60, which were treated with 0.1 µM IDA or 0.1 µM IDA/mPEG-PLGA NPs for 6 hours. Shown is the representative plots (left) and summary of PE⁺ cells (right).

Abbreviations: IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles; PE, phycoerythrin.

Figure 7 Antileukemia activity of IDA and IDA/mPEG-PLGA NPs in leukemia cells in vitro.

Notes: (A) HL-60 and U937 cells were treated with different concentrations of IDA or IDA/mPEG-PLGA for 24 hours. Cell growth was measured by the MTT assay. IC₅₀ was calculated for HL-60 and U937 cells. Apoptosis was measured by annexin V/PI staining in HL-60 (B) and U937 (C) cells, which were treated with 0.01 µM IDA/mPEG-PLGA NPs, 0.01 µM IDA, or mPEG-PLGA NPs for 4 days. *P<0.01.

Abbreviations: IC₅₀, half maximal inhibitory concentration; IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles; PI, propidium iodide.

Figure 8 Antileukemia activity of IDA and IDA/mPEG-PLGA in MLL-AF9-induced murine leukemia in vivo.

Notes: (A) GFP⁺ cells were measured in BM from the MLL-AF9-induced murine leukemia treated with IDA/mPEG-PLGA NPs, IDA, mPEG-PLGA, or vehicle when vehicle mice developed full-blown leukemia (n=4). Shown is the representative plots (left) and summary of GFP⁺ cells (right). **P<0.01. (B) A representative image of blood, BM, and spleen smears in the MLL-AF9-induced leukemia treated with IDA/mPEG-PLGA NPs, IDA, mPEG-PLGA, or vehicle. (C) A representative image of spleen (left) and summary of spleen weight (right) from the MLL-AF9-induced leukemia treated with IDA/mPEG-PLGA NPs, IDA, mPEG-PLGA, or vehicle (n=4). **P<0.01. (D) Overall survival was analyzed in the MLL-AF9-induced leukemia treated with IDA/mPEG-PLGA NPs, IDA, mPEG-PLGA, or vehicle (n=6). **P<0.01.

Abbreviations: BM, bone marrow; IDA, idarubicin; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); NPs, nanoparticles.

Figure S1 (A–C) GPC data for mPEG-PLGA₁, mPEG-PLGA₂, and mPEG-PLGA₃.

Abbreviations: GPC, gel permeation chromatography; mPEG-PLGA, methoxy poly(ethylene glycol)-b-poly(l-lactide-co-glycolide); MW, molecular weight.