De-esterified tragacanth-chitosan nano-hydrogel for methotrexate delivery; optimization of the formulation by Taguchi design

Figures & data

Table 1. The controllable parameters and their variation levels.

Variable	Level 1	Level 2	Level 3
DET solution concentration (w/v%)	0.005	0.01	0.05
Chitosan molecular weight	5	10	>800kDa
MTX/ DET ratio (w/w%)	1:5	1:10	1:15

Table 2. The basic taguchi L₉(3³) orthogonal array and results for individual responses.

No#	DET level	Chitosan level	Drug to polymer ratio level	Size(nm)	PDI	Zeta Potential (mV)	Zeta Potential Deviation
1	1	1	1	139.6	0.095	24.6	89.30
2	1	2	2	148	0.142	17.6	15.2
3	1	3	3	239.4	0.33	25.5	10.5
4	2	1	2	168.4	0.109	26.6	99.4
5	2	2	3	188.7	0.205	25.6	100
6	2	3	1	202.1	0.298	23.9	18.10
7	3	1	3	460	0.266	22.8	120
8	3	2	1	493	0.39	20.7	127
9	3	3	2	452.2	0.366	26.7	7.55

Table 3. Orthogonal array and signal to noise ratio for each response.

No#	DET solution concentration level	Chitosan molecular weight level	MTX/DET level	SNR of Size	SNR of PDI	SNR of Zeta potential	SNR of Zeta deviation
1	1	1	1	−42.8977	20.4455	27.8187	−39.0170
2	1	2	2	−43.4052	16.9542	24.9103	−23.6369
3	1	3	3	−47.5825	9.6297	28.1308	−20.4238
4	2	1	2	−44.5268	19.2515	28.4976	−39.9477
5	2	2	3	−45.5154	13.7649	28.1648	−40.0000
6	2	3	1	−46.1113	10.5157	27.5680	−25.1536
7	3	1	3	−53.2552	11.5024	27.1587	−41.5836
8	3	2	1	−53.8569	8.1787	26.3194	−42.0761
9	3	3	2	−53.1066	8.7304	28.5302	−17.5589

Figure 1. The S/N ratios of controlled parameters for size (A); zeta potential (B); PDI (C); and zeta deviation (D).

Table 4. The ANOVA results for responses.

	Size		PDI		Zeta potential		Zeta Deviation
Parameter	F-value	P-value	F-value	P-value	F-value	P-value	F-value	P-value
Chitosan molecular weight	0.13	.882	2.83	.136	2.02	.213	7.5	.023
DET concentration	28.75	.001	1.84	.238	0.64	.562	0.38	.702
Drug to polymer ratio	0.1	.91	0.33	.732	0.17	.848	0.51	.625

Figure 2. A; the release profile of nanoparticles at optimum conditions. B; the TGA thermograms of chitosan, DET, physical mixture, and drug-loaded nanoparticles. C; the TEM photograph of nanoparticles obtained at optimum condition.

Figure 3. A–D; relative cell viability of free methotrexate (MTX), MTX loaded nanoparticles (MTX loaded DET-CS NPs) evaluated on HT-29 (A and B) and MCF-7 (C and D) cells at 24 (A and C) and 72 h (B and D). Used concentrations were 31, 62.5, 125, 250 and 500 µg/mL for free MTX and MTX loaded NPs, and the concentration of blank nanoparticles was same as ones for the drug-loaded NPs. The relative cell viability read for the control (tissue culture polystyrene from culture plates) after 24, and 72 h of incubation was taken as the reference (100%). Data are expressed as the mean ± SEM of three separate experiments. Significance was calculated by ANOVA (*p ≤ .05).

Figure 4. A & B; the Effect of MTX, MTX loaded DET-CS nanoparticles and blank nanoparticles at different concentration on mitochondrial membrane potential (MMP) collapse after 48 h, as detected by Rhodamine 123 using plate reader in HT-29 (A) and MCF-7 (B). C & D; the cellular uptake of DET-CS nanoparticles after 30 min, 2, 6 and 24 h on HT-29 and MCF-7 (C). The uptake of DET-CS nanoparticles after exposure to uptake inhibitors (sodium azide, nystatin, chlorpromazine, amiloride and at 4 °C) after six h (D). Data are expressed as the mean ± SEM of three separate experiments. Significance was calculated by ANOVA (*p ≤ .05).