Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

Figures & data

Table 1 Independent variables and their coded levels in the Box–Behnken design

Independent variables	Symbols	Coded levels
		−1	0	+1
Drug/phosphatidylcholine (%, w/w)	A	10	15	20
Cholesterol (%, w/v)	B	0.1	0.2	0.3
Sonication time (minutes)	C	2	3	4

Table 2 Dependent variables and their constraints in the Box–Behnken design

Dependent variables	Symbol	Constraints
Particle size	Y1	Minimize
Particle size	Y1	Minimize
PDI	Y2	Minimize
Zeta potential	Y3	>±20
Drug loading	Y4	Maximize

Abbreviation: PDI, polydispersity index.

Table 3 Optimum concentration of independent variables for desired formulation with predicted responses and error

Factors			Predicted responses				Observed responses				Predicted error (%)
A	B	C	Y1	Y2	Y3	Y4	Y1	Y2	Y3	Y4	Y1	Y2	Y3	Y4
10.95	0.30	2.54	101.6	0.28	−29.3	35.3	83	0.2	−27.4	33.2	18.3	28.5	6.48	6.5

Note: Data are shown as mean of three independent experiments. A, drug/phosphatidylcholine (%, w/w); B, cholesterol (%, w/v); C, sonication time (minutes); Y₁, particle size (nm); Y₂, PDI; Y₃, zeta potential (mV); Y₄, drug loading (%).

Abbreviation: PDI, polydispersity index.

Figure 1 Characterization of NLA.

Notes: (A) Size distribution and (B) zeta potential by dynamic light scattering technique, (C) morphology by atomic force microscopy at a scale bar of 1 µm and (D) transmission electron microscopy at a scale bar of 100 nm, and (E) cumulative drug release for 48 hours at pH 5.5 and pH 7.4. All experiments were done in triplicate.

Abbreviation: NLA, nanoliposomal artemisinin.

Figure 2 Toxicity of NLA.

Notes: (A) Ex vivo on murine macrophages by MTT assay after 72 hours incubation at a concentration range of 0.82–200 µg/mL. Serum levels of (B) AST and ALT, (C) ALP, (D) creatinine, and (E) urea in healthy animals after 24 hours of administration of artemisinin, NLA, ENL, and AmB at the highest dose. Data are represented as mean ± SD and are from one of the two independent experiments. ***P<0.0001.

Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AmB, amphotericin B; AST, aspartate aminotransaminase; ENL, empty nanoliposome; NLA, nanoliposomal artemisinin.

Figure 3 In vitro antileishmanial activity.

Notes: (A) Giemsa-stained murine macrophages infected with Leishmania donovani parasites at 48 hours in untreated and treated conditions. (B) Percent L. donovani amastigote infection and (C) percent macrophages infected with L. donovani amastigotes at 48 hours after incubation with different concentrations of artemisinin, NLA, and pentamidine (0–50 µg/mL). (D) Nitric oxide levels in the culture supernatant of uninfected, infected, and treated samples at 48 hours by Griess assay. **P<0.001, ***P<0.0001

Abbreviations: NLA, nanoliposomal artemisinin; ns, not significant.

Figure 4 Antileishmanial activity of nanoliposomes in experimental VL.

Notes: (A) Spleen and liver weight in normal, infected, and treated animals 1 week post-treatment. (B) Percentage inhibition of parasites in the spleen and liver 1 week post-treatment. Data are represented as mean ± SD from one of the two independent experiments having five animals per group. *P<0.05, **P<0.001, ***P<0.0001.

Abbreviations: AmB, amphotericin B; ENL, empty nanoliposome; NLA, nanoliposomal artemisinin; VL, visceral leishmaniasis.

Figure 5 Evaluation of cellular immunity.

Notes: (A) Delayed type hypersensitivity response to freeze thawed leishmanial antigen (800 µg/mL) after 24 hours in age-matched, infected control and animals treated with artemisinin, NLA, ENL, and AmB at the highest dose. (B) Proliferative response by CFSE after 72 hours of in vitro stimulation of total lymphocytes with SLA (10 µg/mL) at the highest dose of artemisinin, NLA, ENL, and AmB. (C) Induction of IL-4, (D) IFN-γ, and (E) IL-10 in the culture supernatant of splenocytes at 72 hours by cytokine bead array at the highest dose of artemisinin, NLA, ENL, and AmB. (F) Parasite-specific antibodies IgG1 and IgG2a 1 week posttreatment with artemisinin, NLA, ENL, and AmB at the highest dose measured by ELISA. Data are represented as mean ± SE from one of the two independent groups having five animals per group. *P<0.05, **P<0.001, ***P<0.0001.

Abbreviations: AmB, amphotericin B; CFSE, carboxy fluorescein succinidimyl ester; ELISA, enzyme-linked immunosorbent assay; ENL, empty nanoliposome; FT, freeze thawed; IFN-γ, interferon-γ; IgG, immunoglobulin G; IL, interleukin; NLA, nanoliposomal artemisinin; SLA, soluble leishmanial antigen.

Figure S1 Box–Behnken or response surface plots showing the effect of independent factors on dependent factors.

Notes: (A) Effect of drug/PC and cholesterol. (B) Drug/PC and sonication time and (C) cholesterol and sonication time on particle size. Particle size is an important parameter or variable for the development of a drug delivery system. Equation 7$\text{Particle size}(Y_1)=135.4+2.5\mathrm{A}+9.75\mathrm{B}-26.45{\mathrm{A}}^2+21.45{\mathrm{B}}^2-24.95{\mathrm{C}}^2$(7) suggests dependence of particle size on independent factors. Negative sign indicates a decrease in the particle size, whereas positive sign in the equation represents an increase in the particle size. An increase in the drug/PC has an unfavorable effect on the particle size and similarly amount of cholesterol directly influences and increases the particle size of the nanoliposomes, while the interaction terms do not have any significant effect. (D) Effect of drug/PC and cholesterol, (E) drug/PC and sonication time, and (F) cholesterol and sonication time on PDI. From the polynomial equation (Equation 8$\text{PDI}\left(Y_2\right)=0.32+0.033625\mathrm{B}+0.042875\mathrm{C}+0.0285\text{BC}+0.04225{\mathrm{A}}^2-0.031{\mathrm{B}}^2$(8) ), positive sign indicates synergistic effect, whereas negative sign represents antagonistic effect. Higher value of PDI suggests nonuniform size distribution of nanoliposomes that may lead to aggregation and hence compromises the stability of nanoparticles. (G) Effect of drug/PC and cholesterol, (H) drug/PC and sonication time, and (I) cholesterol and sonication time on zeta potential. From Equation 9$\text{Zeta potential}\left(Y_3\right)=-35.6-1.05\mathrm{B}-1.625\mathrm{C}-1.625\text{AC}+4.7125{\mathrm{A}}^2+2.9625{\mathrm{B}}^2+5.0125{\mathrm{C}}^2$(9) , it is clear as the levels of cholesterol, sonication time, and interaction terms AC, ie, drug/PC and sonication time, have an inverse relationship with zeta potential. (J) Effect of drug/PC and cholesterol, (K) drug/PC and sonication time, and (L) cholesterol and sonication time on drug loading. The relationship or effect of independent variable on drug loading is shown in the equation and Box–Behnken plots generated, indicating the effect of independent variables on particle size, PDI, zeta potential, and drug loading that are given in (J–L). It is clear from the above equation and response surface plots that drug/PC, amount of cholesterol, and sonication time in the formulation had a significant positive effect on the drug loading, ie, an increase in the levels of these constituents at a time directly influences the amount of drug loading.

Abbreviations: PC, phosphatidylcholine; PDI, polydispersity index.

Figure S2 Contour plots representing percent proliferation of splenocytes after 72 hours of in vitro recall with SLA.

Abbreviations: AmB, amphotericin B; CFSE, carboxy fluorescein succinidimyl ester; ENL, empty nanoliposome; NLA, nanoliposomal artemisinin; SLA, soluble leishmanial antigen; SSC, side scatter.

Table S1 Experimental design generated by Design Expert^® 8.0.7.1 consisting of 17 combinations including five replicates at the central level

Formulation	Drug/phosphatidylcholine (%, w/w, A)	Cholesterol (%, w/v, B)	Sonication time (minutes, C)
NLA1	10	0.1	3
NLA2	20	0.1	3
NLA3	10	0.3	3
NLA4	20	0.3	3
NLA5	10	0.2	2
NLA6	20	0.2	2
NLA7	10	0.2	4
NLA8	20	0.2	4
NLA9	15	0.1	2
NLA10	15	0.3	2
NLA11	15	0.1	4
NLA12	15	0.3	4
NLA13	15	0.2	3
NLA14	15	0.2	3
NLA15	15	0.2	3
NLA16	15	0.2	3
NLA17	15	0.2	3

Abbreviation: NLA, nanoliposomal artemisinin.

Table S2 Responses in dependent variables (particle size, PDI, zeta potential, and drug loading after experimental run)

Sample	Size (nm)	PDI	Zeta potential (mV)	Drug loading (mV)
NLA1	72.4	0.20	−28.2	31.6
NLA2	81.3	0.21	−26.2	41.4
NLA3	95.8	0.31	−29.8	33.4
NLA4	100.9	0.26	−28.5	41.3
NLA5	80.2	0.24	−26.1	28.9
NLA6	84.0	0.23	−21.5	41.4
NLA7	84.9	0.33	−26.9	32.1
NLA8	87.2	0.34	−28.8	42.5
NLA9	76.1	0.26	−25.6	38.6
NLA10	103.2	0.26	−27.2	42.5
NLA11	85.1	0.28	−26.8	41.5
NLA12	91.8	0.39	−30.8	42.4
NLA13	135.3	0.32	−36.2	41.5
NLA14	133.0	0.32	−35.1	40.5
NLA15	135.9	0.33	−36.5	42.4
NLA16	135.0	0.31	−34.6	41.9
NLA17	137.8	0.32	−35.3	41.4

Note: Data are shown as mean of three independent experiments.

Abbreviations: NLA, nanoliposomal artemisinin; PDI, polydispersity index.

Table S3 Summary of results of model analysis, lack of fit, R² analysis for measured response

	Y1		Y2		Y3		Y4
	SS	P > F	SS	P > F	SS	P > F	SS	P > F
Source model analysis
Mean vs total	173,821.2		1.420435		14,924.33		26,044.54
Linear vs mean	815	0.7457	0.023792	0.0083	33.325	0.51687	218.8075	8.574969
2FI vs linear	105	0.9880	0.003888	0.4378	12.6075	0.157525	4.1625	0.13039
Quadratic vs 2FI	8,377.565	<0.0001	0.012382	0.0001	262.3128	136.9269	99.78168	35.11938
Cubic vs quadratic	33	0.1376	0.000553	0.1198	2.17	1.257971	4.6575	3.149087
Residual	13.2		0.0002		2.3		1.972
Total	183,165		1.46125		15,237.05		26,373.92
Lack of fit
Linear	8,515.565	<0.0001	0.016823	0.0017	108.6017	0.0038	277.0903	0.0008
2FI	8,410.565	<0.0001	0.012936	0.0014	104.4392	0.0020	264.4828	0.0004
Quadratic	33	0.1376	0.000553	0.1198	4.6575	0.1484	2.17	0.4007
Cubic	0		0		0		0
Pure error	13.2		0.0002		1.972		2.3
R^2 analysis	R^2	Ra^2	PRESS	R^2	Ra^2	PRESS	R^2	Ra^2	PRESS	R^2	Ra^2	PRESS
Linear	0.087224	−0.12342	12,173.25	0.582918	0.486668	0.030825	0.106567	−0.09961	410.732	0.664299	0.586829	201.8112
2FI	0.098461	−0.44246	19,668.79	0.678165	0.485063	0.047497	0.146883	−0.36499	646.9106	0.676936	0.483098	423.4691
Quadratic	0.995056	0.988698	548.625	0.981545	0.957817	0.009165	0.985706	0.967328	38.31375	0.979873	0.953995	77.60125
Cubic	0.998587	0.994349	ND	0.9951	0.980399	ND	0.992645	0.97058	ND	0.994013	0.976052	ND

Abbreviations: PRESS, predicted residual sum of squares; SS, sum of squares; ND, not determined.

Table S4 Storage stability at different time points at 37°C for 25 days

Parameter	Initial	After 7 days	After 15 days	After 25 days
Particle size (nm)	83.13±16.0	82.67±4.1	81.35±2.81	80.70±1.90
PDI	0.21±0.03	0.21±0.025	0.20±0.029	0.19±0.006
Zeta potential (mV)	−27.36±5.7	−27.16±0.85	−27.06±1.30	−27.46±1.95
Drug loading (%)	33.23±2.10	31.41±2.43	29.87±2.66	32.64±2.19

Note: Data are shown as mean ± SD and are representative of three independent experiments.

Abbreviation: PDI, polydispersity index.

Table S5 Mechanism of drug release under in vitro conditions at endosomal and physiological pH at 37°C

Model	Plot		R^2 values
	X-axis	Y-axis	pH 5.5	pH 7.4
Zero-order model	Fraction of drug released	Time	0.903	0.897
First-order model	Log% drug remaining	Time	0.987	0.988
Higuchi model	Fraction of drug released	√Time	0.996	0.995
Hixson–Crowell model	Cube root of fraction drug remaining	Time	0.971	0.970
Korsmeyer–Peppas model	Log fraction of drug released	Log time	0.579	0.546

Note: Data are shown as mean of three independent experiments.

Table S6 IC₅₀ values of NLA and artemisinin in an ex vivo model of leishmaniaisis

Parasites/macrophages	Artemisinin	NLA
Intracellular amastigotes	15.2±2.5	6.0±1.4
Infected macrophages	12.7±2.5	5.1±0.9

Note: Data are shown as mean ± SD and are representative of one from two independent experiments.

Abbreviation: NLA, nanoliposomal artemisinin.