Critical physicochemical and biological attributes of nanoemulsions for pulmonary delivery of rifampicin by nebulization technique in tuberculosis treatment

Figures & data

Table 1. Composition of the nanoemulsion formulations.

Composition (% w/w)
	First-generation nanoemulsion					Second-generation nanoemulsion			Third-generation nanoemulsion
	L1	L2	L3	L4	L5	L6	L7	L8	L9
Oleic acid	5.00	5.00	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Rifampicin	0.88	0.88	0.88	0.88	0.88	0.88	0.88	0.88	0.88
Ethanol	1.00	1.00	1.00	5.00	10.00	1.00	1.00	1.00	1.00
Tween-80	5.00	10.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00
Chitosan	0.00	0.00	0.00	0.00	0.00	0.10	0.25	0.50	0.00
Chitosan-folate conjugate	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.25
Normal saline	88.13	83.13	78.13	74.13	69.13	78.03	77.88	77.63	77.88

Figure 2. Profiles of (a) average droplet size and (b) polydispersity index of L1 to L5, (c) size distribution and (d) TEM image of L3, ESEM images of (e) first (L3), (f) second (L7) and (g) third (L9) generation nanoemulsions (magnification factor: 16,000 ×).

Table 2. Physicochemical characteristics of nanoemulsions with reference to (a) average droplet size, PDI and zeta potential, and (b) pH, surface tension, density, specific viscosity, refractive index, drug content, burst drug release extent and duration to complete drug dissolution.

(a)
Formulation	Average Size (nm)	PDI	Zeta potential (mV)
L3	43.89 ± 0.36	0.16 ± 0.03	–2.50 ± 1.06
L6	50.70 ± 0.64	0.26 ± 0.03	–1.68 ± 0.12
L7	52.12 ± 0.36	0.25 ± 0.03	4.18 ± 0.13
L8	108.50 ± 0.01	0.46 ± 0.05	2.16 ± 0.50
L9	59.69 ± 0.26	0.23 ± 0.01	0.70 ± 0.24
(b)
Characteristics	First-generation nanoemulsion (L3)	Second generation nanoemulsion (L7)	Third-generation nanoemulsion (L9)
pH	4.90 ± 0.02	5.06 ± 0.02	4.59 ± 0.04
Surface tension (mN/m^2)	33.50 ± 0.50	35.00 ± 0.29	36.33 ± 0.94
Density (g/cm^3)	1.00 ± 0.00	1.01 ± 0.00	1.01 ± 0.00
Specific viscosity	0.16 ± 0.02	0.32 ± 0.01	0.22 ± 0.01
Refractive index	1.34 ± 4.70 × 10^−6	1.34 ± 4.70 × 10^−6	1.34 ± 4.70 × 10^−6
Drug content (mg/ml)	8.51 ± 0.28	8.18 ± 0.35	8.11 ± 0.04
Burst drug release extent (%)	54.82 ± 12.01	38.69 ± 5.71	64.38 ± 12.36
T100%	6	24	28

Burst drug release extent denoted the fraction of drug released within the first 0.17 h of dissolution.

T_100% denoted the duration required to achieve 100% drug dissolution.

Figure 3. (a) Cumulative percentage drug release profiles and (b) In vitro Anderson cascade impaction deposition of drug from first, second and third-generation nanoemulsions.

Table 3. Profiling of (a) aerosolization and inhalation performances of nanoemulsions determined using the Anderson cascade impaction and gravimetric methods, and (b) their relationship with the physicochemical properties of nanoemulsions.

(a)Parameter	First-generation nanoemulsion (L3)	Second-generation nanoemulsion (L7)	Third-generation nanoemulsion (L9)
Total Aerosol Output (%)	99.27 ± 0.41	98.82 ± 0.35	98.53 ± 0.74
Aerosol output rate (g/min)	0.19 ± 0.01	0.17 ± 0.01	0.15 ± 0.01
TD (mg)	2.63	2.63	2.63
ED (mg)	2.49 ± 0.17	2.44 ± 0.09	2.35 ± 0.05
DD (mg)	2.10 ± 0.20	2.00 ± 0.15	2.00 ± 0.08
PD (%)	94.68 ± 6.64	93.04 ± 3.62	90.00 ± 1.81
PI (%)	79.85 ± 7.63	76.04 ± 5.63	75.93 ± 3.18
MMAD (µm)	3.02 ± 0.19	3.21 ± 0.67	3.84 ± 0.38
GSD	2.03 ± 0.13	2.12 ± 0.20	2.24 ± 0.02
FPD 0–f (mg)	2.10 ± 0.20	2.00 ± 0.15	1.99 ± 0.08
FPF	84.38 ± 6.14	81.70 ± 4.47	84.67 ± 2.52
RF	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0
FPD<5µm (mg)	1.82 ± 0.22	1.65 ± 0.21	1.47 ± 0.12
FPF	73.15 ± 5.17	67.71 ± 10.23	62.37 ± 4.58
RF	86.79 ± 4.52	82.75 ± 10.68	73.60 ± 3.27
FPD<3µm (mg)	1.66 ± 0.23	1.45 ± 0.31	1.15 ± 0.14
FPF	66.75 ± 5.31	59.79 ± 14.61	48.88 ± 5.58
RF	79.26 ± 6.35	72.97 ± 16.58	57.64 ± 4.95
(b)
Total aerosol output	r	p	Aerosol output rate	r	p
Size	–0.995	.064**	Size	–1.000	.017*
PDI	–0.818	.390	PDI	–0.773	.437
pH	0.549	.630	pH	0.610	.583
Zeta potential	–0.584	.603	Zeta potential	–0.522	.650
Specific viscosity	–0.499	.667	Specific viscosity	–0.434	.714
Surface tension	–0.996	.056**	Surface tension	–1.000	.010*
Refractive index	–0.556	.624	Refractive index	–0.616	.578
PD	r	p	PI	r	p
Size	–0.981	.124	Size	–0.889	.302
PDI	–0.625	.571	PDI	–0.975	.144
pH	0.768	.443	pH	0.204	.869
Zeta potential	–0.323	.791	Zeta potential	–0.841	.364
Specific viscosity	–0.225	.855	Specific viscosity	–0.782	.429
Surface tension	–0.979	.131	Surface tension	–0.894	.296
Refractive index	–0.724	.485	Refractive index	–0.139	.911
FPF<5 µm	r	P	FPF<3 µm	r	p
Size	–1.0	.012*	Size	–0.989	.096**
PDI	–0.752	.458	PDI	–0.658	.542
pH	0.643	.555	pH	0.739	.471
Zeta potential	–0.484	.679	Zeta potential	–0.364	.763
Specific viscosity	–0.393	.743	Specific viscosity	–0.268	.827
Surface tension	–1.000	.019*	Surface tension	–0.987	.103
Refractive index	–0.592	.597	Refractive index	–0.693	.513
RF<5 µm	R	P	RF<3 µm	r	p
Size	–0.970	.155	Size	–0.966	.166
PDI	–0.586	.602	PDI	–0.572	.612
pH	0.788	.412	pH	0.808	.401
Zeta potential	–0.276	.822	Zeta potential	–0.26	.833
Specific viscosity	–0.177	.886	Specific viscosity	–0.161	.897
Surface tension	–0.968	.162	Surface tension	–0.963	.173
Refractive index	–0.757	.453	Refractive index	–0.768	.443

Level of significance *p < .050, **p < .100.

Table 4. Aerosolization profiles of nanoemulsions determined by laser diffraction method.

Parameter	First generation nanoemulsion (L3)	Second generation nanoemulsion (L7)	Third-generation nanoemulsion (L9)
Dv(10) (µm)	2.33 ± 0.05	2.56 ± 0.07	2.63 ± 0.25
Dv(50) (µm)	4.95 ± 0.07	5.24 ± 0.12	6.34 ± 0.56
Dv(90) (µm)	9.59 ± 0.15	10.28 ± 0.24	14.46 ± 1.83
D[3,2] µm	3.52 ± 0.10	4.49 ± 0.04	4.03 ± 0.12
D[4,3] µm	5.51 ± 0.07	5.93 ± 0.06	7.61 ± 0.32
SSA (m^2/cc)	1.70 ± 0.04	1.34 ± 0.01	1.49 ± 0.33
GSD/Span	1.87 ± 0.09/1.47 ± 0.01	1.64 ± 0.06/1.47 ± 0.01	2.08 ± 0.08/1.86 ± 0.01
FPF<3µm	18.23 ± 0.31	15.09 ± 0.39	12.72 ± 0.87
FPF<5µm	45.47 ± 0.59	41.50 ± 0.72	31.88 ± 1.80

Figure 4. Profiles of (a) average corrected cell fluorescence intensity within alveolar macrophages after treatment with second and third-generation nanoemulsions, (b) confocal laser scanning microscopic images showing endocytosis of second generation nanoemulsion (i–iii) and third-generation nanoemulsion (iv–vi) in macrophages where (i) and (iv) were fluorescent micrographs, (ii) and (v) were differential interference contrast images, (iii) and (vi) were merge images of fluorescent and differential interference contrast images, (c) average viability of alveolar macrophages following treatment with rifampicin solution and rifampicin loaded nanoemulsions.

Figure 5. Profiles of (a) average plasma rifampicin concentration-time curves, (b) rifampicin concentrations in homogenized lung and (c) pharmacokinetics parameters obtained after intratracheal administration of rifampicin loaded nanoemulsions at dose of 2 mg/kg body weight.