Comparative analysis between four model nanoformulations of amphotericin B-chitosan, amphotericin B-dendrimer, betulinic acid-chitosan and betulinic acid-dendrimer for treatment of Leishmania major: real-time PCR assay plus

Figures & data

Table 1 Binding energy (Kcal.mol⁻¹) in the four nanodrugs complexes at the optimized stable condition

Complex	Binding energy
BD	−4.48
AD	−5.27
BK	−5.00
AK	−5.08

Figure 1 The results of molecular modeling and docking calculations for the four different nanodrugs.

Table 2 The results of size, size distribution (PDI) and zeta potential of different nanoparticles

Parameters Nanoparticle	Size (nm)	PDI	Zeta potential (mV)
K	102±10	0.2±0.1	14±2
BK	124±14	0.3±0.1	6.5±1
AK	112±15	0.45±0.15	8±1.5
D	90±13	0.25±0.17	−5.8±0.9
BD	156±15	0.31±0.14	−14.6±1.7
AD	138±11	0.11±0.13	−18.8±2.5

Figure 2 The mean cumulative release of Amp and BA from the four nanodrugs of AK, BK, AD and BD after 48 h (slow release).

Figure 3 The flow cytometry results related to cellular uptake of nanodrug AK (nano amphotericin chitosan) =98.6%, BK=98%, AD=64% and BD=94.6. Also, the cellular uptake of A was 30% and B was equal to 28%.

Figure 4 The viability effects of the nanodrugs AK, BK, AD and BD at the same concentration of 20 µg/mL along with K(Chitosan) and D(dendrimer) at the concentration of 10 µg/mL compared to control group on the Balb/c mouse-derived peritoneal macrophages after 48 h incubation. The values are expressed as Mean ± SD from three independent experiments with P˂0.001.

Figure 5 The killing effects of nanodrugs BK(nano amphotericin chitosan)20 µg/mL (with the killing effects of 88%), AK20 µg/mL, AD20 µg/mL, BD20 µg/mL, nano K(Chitosan)10 µg/mL, A20 µg/mL and B20 µg/mL on the viability of L. major promastigotes after 48 h incubation. The values are expressed as Mean ± SD from three independent experiments with P˂0.001.

Figure 6 The therapeutic effects of nanodrugs AK(nano amphotericin chitosan)20 µg/mL, AD20 µg/mL, BK20 µg/mL and BD20 µg/mL on the L. major-infected macrophages after 48 h incubation. The highest killing effects were related to BK20 µg/mL with the killing effects of 81%. The values are expressed as Mean ± SD from three independent experiments with P˂0.001.

Figure 7 The lesion size in L. major-infected mice received various formulations including K12.5, D35.5, AD50, B(20 and 40), GUL200, AK10, BD40, A (10 and 50) and BK20 mg/kg. As the figure shows, the lesion size reached to zero in nanodrugs of AK10 mg/kg and BK20 mg/kg receivers (P˂0.001).

Figure 8 The evaluation results of parasite burden in mice groups of BK20, AK‎10, BD40, AD50, A(10 and 50), B(20 and 40), GUL200, K12.5, D35.5 mg/kg and control group. As the results showed the lowest parasite burden was related to BK20=5.57×10⁶±0.2 and AK10 mg/kg=8.22×10⁶±0.3 groups. The results are expressed as Mean ± SD from three independent experiments.

Figure 9 The standard curve obtained from the amplification of L. major SODB1 gene over 5-fold serial dilutions of the parasitic DNA, ranging from 5.3×10⁴ parasites to 5.3 parasites per reaction.

Table 3 Calculation of the parasite number of popliteal lymph nodes in different groups of L. major infected BALB/C mice at 8 weeks post infection using real-time PCR and LDA

Groups	Number of parasites/half of the popliteal lymph node (LDA)	Mean CT ± SD	Number of parasites/2×10^6 cells of half of popliteal lymph node (real-time PCR)
Dendrimer	12.24×10^6	26.10	10,000
Chitosan nanoparticles	11.35×10^6	26.49	8000
Glucantime	10.75×10^6	26.55	7600
Betulinic acid	10.47×10^6	26.54	7400
Amphotericin B	10.44×10^6	26.60	7200
AD50 mg/kg	10.36×10^6	26.70	6800
BD40 mg/kg	9.44×10^6	26.80	6400
AK10 mg/kg	8.22×10^6	27.30	5400
BK20 mg/kg	5.47×10^6	27.61	4600

Table 4(A) Synthesis and characterization of various types of chitosan-based nanoparticles in vitro and in vivo environments, reported in different studies

Reference Properties	^Citation48	^Citation5	^Citation27	^Citation24	^Citation7	Present study
Name	NQC-AmpB	HePC-AmB-CNLCs	Cs-PF-AmB-M	Chitosan-coated AmB-loaded SLNs	MTC AmB	AK
Nanoparticle size (nm)	135	–	–	148	–	102
Nanodrugs size (nm)	155	150	156	166	362	112
Synthesis method	Ionic gelation	Emulsification and homogenization	Ionic gelation	Emulsification and solvent evaporation	Ionic gelation	Phase separation
Drug loading efficiency (%) and methods used for confirmation	81%; DLS, SEM, TEM	80%; DLS, TEM	63%; DLS	82%; SEM, DLS	73%; FTIR, SEM	90%; DLS, SEM, TEM, AFM, HNMR, FTIR
Pattern of drug release	Controlled release	Controlled release	Controlled release	Biphasic	Controlled release	Controlled release
Cellular uptake (%)	32%	55%	22%	34%	45%	98.6%
In vitro viability (%) (MTT)	50% at 9 µg/mL	58% at 10 µg/mL	72% at 20 µg/mL	49% at 20 µg/mL	34% at 57 µg/mL	100% at 20 µg/mL
Promastigote killing (%)	50% at 1 µg/mL	–	–	–	–	82% at 20 µg/mL
Amastigote inhibition (%)	61%	68%	50%	50%	61%	78%
Leishmania species	Amazonensis	Donovani	Donovani	Donovani	Donovani	Major

Table 4(B) Synthesis and characterization of various types of dendrimer nanoparticles in vitro and in vivo environments, reported in different studies

Reference properties	^Citation7	^Citation6	^Citation30	Present study
Name	PADRE-PDD-LAmB	AmB-PPI dendrimer	MdPPIA	AD	BD
Size of dendrimer (nm)	-	93	80	90	90
Size of drug loaded dendrimer (nm)	142	138	122	138	156
Synthesis method	Divergent	Divergent	Divergent	Divergent	Divergent
Drug loading efficiency (%) and its confirmation method	58%; TEM, DLS	55%; flowcytometry and fluorescent microscopy	76%; FTIR, DLS	84%; AFM, DLS, FTIR, HNMR	96%; AFM, DLS, FTIR, HNMR
Pattern of drug release	–	Controlled release	Biphasic	Controlled release	Controlled release
Cellular uptake (%)	–	42%	76%	64%	94.6%
In vitro viability (%) (MTT)	75% at 1 µg/mL	56% at 33 µg/mL	55% at 1, 5, 10, 25 µg/mL	100% at 20 µg/mL	100% at 20 µg/mL
Promastigote killing (%)	–	–	–	61% at 20 µg/mL	73% at 20 µg/mL
Amastigote inhibition (%)	–	23%	32%	52%	61%
Leishmania species	Donovani	Donovani	Donovani	Major	Major

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