Cooperation of IRAK1/4 inhibitor and ABT-737 in nanoparticles for synergistic therapy of T cell acute lymphoblastic leukemia

Figures & data

Table 1 Selective variables level in Box-Behnken design

Variables	Factors	Code
		−1	0	1
A	Polymer concentration (%)	0.5	2	3.5
B	Oil phase:water phase (V/V)	1:1	1:7	1:13
C	Emulsifier concentration (%)	0.5	2	3.5

Table 2 Box-Behnken design and matrix and corresponding responses

Run	Variables			Responses
	A	B	C	Y (%)
1	0	0	0	7.5
2	−1	1	0	2.7
3	−1	−1	0	0.9
4	1	0	−1	5.4
5	−1	0	1	1.5
6	0	1	−1	5.5
7	0	−1	−1	5.3
8	1	1	0	5.6
9	1	0	1	6.4
10	1	−1	0	5.7
11	0	1	1	6.5
12	0	0	0	7.4
13	0	−1	1	5.3
14	−1	0	−1	1.4
15	0	0	0	7.3

Notes: Y is drug loading (%), and A, B and C are coded values for polymer concentration (%), oil phase:water phase (V/V), and emulsifier concentration (%), respectively.

Table 3 ANOVA results for response Y (drug loading)

Source	df	Adj SS	Adj MS	F-value	p-value
Model	9	70.0235	7.7804	183.07	0
Linear	3	36.1975	12.0658	283.9	0
A	1	34.445	34.445	810.47	0
B	1	1.2013	1.2013	28.26	0.003
C	1	0.5513	0.5513	12.97	0.016
Square	3	32.471	10.8237	254.67	0
A*A	1	29.4669	29.4669	693.34	0
B*B	1	2.6677	2.6677	62.77	0.001
C*C	1	2.9908	2.9908	70.37	0
2-way interaction	3	1.355	0.4517	10.63	0.013
A*B	1	0.9025	0.9025	21.24	0.006
A*C	1	0.2025	0.2025	4.76	0.081
B*C	1	0.25	0.25	5.88	0.06
Error	5	0.2125	0.0425
Lack-of-fit	3	0.1925	0.0642	6.42	0.138
Pure error	2	0.02	0.01
Total	14	70.236

Notes: R²=0.9970, adjusted R²=0.9915, and predicted R²=0.9555. Y is drug loading (%), and A, B and C are coded values for polymer concentration (%), oil phase:water phase (V/V), and emulsifier concentration (%), respectively.

Abbreviations: df, degrees of freedom; Adj SS, adjusted sum of squares; Adj MS, adjusted mean square.

Table 4 Predicted and experimental response values of optimized condition

Response	Predicted value	Experimental value	Prediction error (%)
Y	7.31	7.4	0.12

Notes: Prediction error, relative standard deviation <2%. Y is drug loading (%).

Figure 1 Three-dimensional response surface plots and contour plots for Y as a function of two of three variables at a fixed value of the third one variable.

Notes: Response surface plot for Y as a function of (A) polymer concentration and volume ratios of oil phase to water phase at fixed emulsifier concentration, (B) polymer concentration and emulsifier concentration at fixed volume ratios of oil phase to water phase, (C) volume ratios of oil phase to water phase and emulsifier concentration at fixed polymer concentration, (D–F) contour plots for Y as a function of two of three variables at a fixed value of the third one variable as (A–C). Y is drug loading (%), and A, B and C are coded values for polymer concentration (%), oil phase:water phase (V/V), and emulsifier concentration (%), respectively.

Figure 2 Characterization of IRAK/ABT-NP.

Notes: (A) SEM image of IRAK/ABT-NP, (B) size and size distribution of IRAK/ABT-NP, (C) TEM image of IRAK/ABT-NP. (D) Confocal laser scanning microcopy images of fluorescently labeled IRAK1/4 inhibitor and ABT-737 co-encapsulated in IRAK/ABT-NP, (E) In vitro release of drugs from IRAK/ABT-NP.

Abbreviations: IRAK/ABT-NP, IRAK1/4 inhibitor and ABT-737 co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid) nanoparticles; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Figure 3 (A) Representative fluorescence images of uptake of IRAK/ABT-NP in Jurkat cells compared to the IRAK1/4 and ABT-737 combined solution, (B) fluorescence intensity analysis of images from (A).

Abbreviation: IRAK/ABT-NP, IRAK1/4 inhibitor and ABT-737 co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid) nanoparticles.

Figure 4 In vitro cell viability of Jurkat cells treated with IRAK/ABT-NP, IRAK1/4 inhibitor alone, ABT-737 alone and combined solution at different concentrations.

Abbreviation: IRAK/ABT-NP, IRAK1/4 inhibitor and ABT-737 co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid) nanoparticles.

Figure 5 Annexin V/7-AAD staining used for flow cytometry analysis of apoptosis in Jurkat cells induced by IRAK/ABT-NP compared to IRAK1/4 inhibitor alone, ABT-737 alone and combined solution.

Abbreviation: IRAK/ABT-NP, IRAK1/4 inhibitor and ABT-737 co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid) nanoparticles.

Figure 6 (A) Representative images of peripheral blood smears from T-ALL xenograft mice after treatment with IRAK/ABT-NP, IRAK1/4 inhibitor alone, ABT-737 alone and combined solution. (B) Changes of WBC in T-ALL xenograft mice after treatment with drugs in different formulations. (C) Survival curves in each treated group of mice.

Abbreviations: T-ALL, T cell acute lymphoblastic leukemia; IRAK/ABT-NP, IRAK1/4 inhibitor and ABT-737 co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid) nanoparticles; WBC, white blood cells.