Nanostructured lipid carriers for incorporation of copper(II) complexes to be used against Mycobacterium tuberculosis

Figures & data

Figure 1 Determination of the mean hydrodynamic diameter (Z-Ave) (d.nm) and polydispersity index versus time in days (mean ± standard deviation; n=3).

Note: ^a–dDifferent letters in each column indicate that the differences between means were statistically significant (P<0.05).

Figure 2 Values of zeta potential (mV) versus time in days (mean ± standard deviation; n=3).

Note: ^a–cDifferent letters in each column indicate a statistically significant difference between means (P<0.05).

Abbreviation: ZP, zeta potential.

Table 1 NLC components

NLCs	E-40	Composition (%)			CTAB	Water (Milli-Q)
		CCTG	PEG-40	P-407
F1	2.07	2.05	0.88	1.00	–	94.00
F2	2.07	2.05	0.88	2.00	–	93.00
F3	2.07	2.05	0.88	2.50	–	92.50
F4	2.07	2.05	0.88	3.00	–	92.00
F5	2.07	2.05	0.88	3.50	–	91.50
F6	2.07	2.05	0.88	4.00	–	91.00
F7	2.07	2.05	0.88	1.00	0.50	93.50
F8	2.07	2.05	0.88	2.00	0.50	92.50
F9	2.07	2.05	0.88	2.50	0.50	92.00
F10	2.07	2.05	0.88	3.00	0.50	91.50
F11	2.07	2.05	0.88	3.50	0.50	91.00
F12	2.07	2.05	0.88	4.00	0.50	90.50

Note: Column 1 contains the respective carrier formulations with the respective compositions studied.

Abbreviations: NLC, nanostructured lipid carrier; CCTG, caprylic/capric triglyceride; PEG-40, polyoxyl 40 hydrogenated castor oil; P-407, poloxamer 407; CTAB, cetyltrimethylammonium bromide.

Table 2 Values expressed as mean ± SD of the mean hydrodynamic diameter and PDI of the NLCs developed (n=3)

	Mean Z-Ave (d.nm) ± SD		Mean PDI ± SD
	1 day	30 days	1 day	30 days
F1	90.06±0.99	106.10±0.77	0.28±0.02	0.18±0.01
F2	102.20±1.47	112.40±1.05	0.35±0.05	0.19±0.01
F3	87.54±0.89	108.10±1.25	0.30±0.02	0.18±0.01
F4	103.50±1.06	118.80±1.86	0.26±0.01	0.15±0.01
F5	88.24±0.13	108.80±1.62	0.27±0.01	0.14±0.01
F6	96.83±0.77	111.70±1.31	0.28±0.01	0.14±0.01
F7	49.50±0.19	75.94±1.41	0.19±0.01	0.32±0.01
F8	77.24±1.76	70.08±0.29	0.41±0.01	0.22±0.01
F9	83.76±1.52	83.88±0.08	0.42±0.02	0.28±0.01
F10	70.59±0.33	82.47±1.16	0.32±0.03	0.24±0.01
F11	83.36±0.24	93.75±0.56	0.26±0.01	0.25±0.01
F12	83.57±5.63	92.38±1.44	0.23±0.01	0.22±0.01

Abbreviations: NLCs, nanostructured lipid carriers; SD, standard deviation; PDI, polydispersity index.

Figure 3 Two-dimensional images of photomicrographs of NLCs: (A) F11, (B) F11.1, (C) F11.2, and (D) F11.3.

Abbreviation: NLCs, nanostructured lipid carriers.

Figure 4 DSC scans of free copper(II) complexes 1, 2, and 3 and the components used in preparation of NLCs: (A) polyoxyl-40 hydrogenated castor oil (PEG-40); (B) cetyltrimethylammonium bromide (CTAB); (C) [CuCl₂(INH)₂]·H₂O (1); (D) [Cu(NCS)₂(INH)₂]·5H₂O (2); (E) [Cu(NCO)₂(INH)₂]·4H₂O (3); (F) poloxamer 407 (P-407); (G) polyoxyethylene 40 stearate (E-40); (H) caprylic/capric triglyceride (CCTG).

Abbreviations: CTAB, cetyltrimethylammonium bromide; DSC, differential scanning calorimetry; NLCs, nanostructured lipid carriers; Endo, endhotermic.

Table 3 Melting points of the NLC components: E-40, CCTG, PEG-40, P-407, and CTAB

Components	MP (°C)
E-40	52.45
CCTG	−5.67
PEG-40	16.87
P-407	55.67
CTAB	103.93

Abbreviations: NLC, nanostructured lipid carrier; E-40, polyoxyethylene 40 stearate; CCTG, caprylic/capric triglyceride; PEG-40, polyoxyl 40 hydrogenated castor oil; P-407, poloxamer 407; CTAB, cetyltrimethylammonium bromide; MP, melting point.

Figure 5 DSC scans of the F11 NLC and copper(II) complex-loaded NLCs, (A) F11, (B) F11.1, (C) F11.2, and (D) F11.3.

Abbreviations: DSC, differential scanning calorimetry; NLCs, nanostructured lipid carriers; Endo, endhotermic.

Table 4 In vitro antimicrobial activities of the free copper(II) complexes and those loaded into the NLCs expressed as the MIC values (μg/mL and μmol/L)

Complexes	MIC (μg/mL)	MIC (μmol/L)
1	103.63±37.07	253.0±90.70
2	63.26±36.52	116.0±67.12
3	80.71±38.47	163.0±77.89
F11	2.47±1.01	2.47±1.01
F11.1	1.87±0.12	4.57±0.29
F11.2	2.31±1.42	4.24±2.61
F11.3	1.96±1.32	3.97±2.67
Rifampicin	0.400±0.01	0.522±0.01

Abbreviations: NLCs, nanostructured lipid carriers; MIC, minimum inhibitory concentration.

Figure 6 Percentage survival of Swiss mice over 14 days after a single administration of free copper(II) complexes 1, 2, and 3 and of those administered copper(II) loaded into the F11 NLC and SO, via gavage at a dose of 1,000 mg/kg bodyweight.

Abbreviations: NLC, nanostructured lipid carrier; SO, sunflower oil.

Figure 7 Percentage survival of Swiss mice over 14 days after a single administration of free copper(II) complex 1 via gavage at a dose of 500 mg/kg bodyweight.

Figure 8 Percentage survival of Swiss mice over 14 days after a single administration of free copper(II) complex 1 via gavage at a dose of 300 mg/kg bodyweight.

Figure 9 Quantification of the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity levels in Swiss mice in terms of units per liter (U/L). The differences between the means were not statistically significant (P>0.05).

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; SO, sunflower oil; U/L, units per liter.