Nanophyto-gel against multi-drug resistant Pseudomonas aeruginosa burn wound infection

Figures & data

Table 1. Compositions of NLC formulations.

Formula	Precirol (mg)	Stearic acid (mg)	Labrafac (mg)	Cinnamon oil (mg)	Tween 80 (mg)	Water (g)
1	833	0	0	167	300	8.7
2	1667	0	166	167	50	8
3	500	0	333	167	300	8.7
4	1667	0	166	167	300	7.7
5	500	0	333	167	50	9
6	1000	0	833	167	50	8
7	1000	0	833	167	300	7.7
8	833	0	0	167	50	9
9	0	500	333	167	300	8.7
10	0	1667	166	167	300	7.7
11	0	1000	833	167	50	8
12	0	500	333	167	50	9
13	0	833	0	167	300	8.7
14	0	1000	833	167	300	7.7
15	0	1667	166	167	50	8
16	0	833	0	167	50	9

Table 2. 2⁴ full factorial design variables and levels of NLC-cinnamon oil.

Type of variables	Levels
Factors (independent variables)	Lower limit	Upper limit
A: type of solid lipid	Precirol	Stearic acid
B: ratio of solid to liquid lipid	1:1 (min)	5:1 (max)
C: total lipids (%w/w)	10 (min)	20 (max)
D: surfactant concentration (%w/w)	0.5 (min)	3 (max)
Responses (dependent variables)	Desirability constraints
X1: particle size (nm)	Minimize
X2: zeta potential (mV)	Maximize (as an absolute value)

Table 3. Antibiogram of used multidrug resistant P. aeruginosa strains.

Organisms	No. of isolates	The used antibiotics and percent of resistance pattern
		Gentamycin (GN)%	Amikacin (AK)%	Ampicillin/ Sulbactam (SAM)%	Amoxicillin/ Clavulanate (AMC)%	Cefepime (FEP)%	Cefoperazone (CFP)%	Cefotaxime (CTX)%	Ceftriaxone (CRO)%	Piperacillin (PIP)%	Imipenem (IPM)%	Meropenem (MEM)%	Ciprofloxacin (CIP)%	Levofloxacin (LEV)%	Trimethoprim/ Sulfamethoxazole (SXT)%	Aztreonam (ATM)%	Ceftazidime (CAZ)%	Chloramphenicol (C)%	Colistin (CL)%	Tetracycline (TE)%
P. aeruginosa	40	55.8	55.8	74.4	74.4	55.8	55.8	100	–	74.4	55.8	55.8	55.8	55.8	–	55.8	74.4	55.8	55.8	–

Table 4. The factorial design formulation matrix (n = 3).

Random run	Factor 1 (A) Types of solid lipid	Factor 2 (B) Solid:liquid lipid ratio	Factor 3 (C) Total lipid concentration % w/v	Factor 4 (D) Surfactant concentration % w/v	Response 1 (X1) Particle size, nm	Response 2 (X2) Zeta potential, mV
1	Stearic acid	5:1	10.00	3.00	120.00 ± 3.522	−37.60 ± 3.66
2	Precirol	1:1	10.00	0.50	212.70 ± 12.08	−25.20 ± 2.71
3	Precirol	5:1	10.00	3.00	677.10 ± 80.86	−38.70 ± 6.33
4	Stearic acid	5:1	10.00	0.50	130.30 ± 4.996	−34.70 ± 3.38
5	Stearic acid	1:1	20.00	0.50	173.60 ± 8.795	−39.00 ± 4.02
6	Stearic acid	1:1	20.00	0.50	104.70 ± 3.584	−30.80 ± 4.31
7	Precirol	1:1	10.00	3.00	169.50 ± 1.041	−39.10 ± 6.00
8	Stearic acid	5:1	10.00	3.00	140.50 ± 0.473	−35.60 ± 3.24
9	Precirol	1:1	20.00	0.50	220.50 ± 3.635	−35.20 ± 5.12
10	Stearic acid	5:1	20.00	3.00	109.70 ± 1.250	−43.10 ± 7.06
11	Precirol	5:1	20.00	0.50	157.20 ± 5.36	−23.80 ± 1.96
12	Precirol	1:1	20.00	3.00	236.00 ± 1.803	−34.60 ± 2.68
13	Precirol	5:1	20.00	0.50	141.50 ± 2.650	−11.30 ± 3.99
14	Stearic acid	1:1	10.00	0.50	172.30 ± 2.804	−43.50 ± 6.39
15	Precirol	3:1	15.00	3.00	136.50 ± 0.101	−35.50 ± 3.87
16	Stearic acid	5:1	20.00	3.00	113.60 ± 1.589	−30.30 ± 2.07

Figure 1. 3D plot model graphs demonstrating the effect of factors. (A) Type of solid lipid, (B) solid:liquid lipid ratio, (C) total lipid content, and (D) SAA concentration on mean particle size (nm).

Figure 2. 3D plot model graphs demonstrating the effect of factors. (A) Type of solid lipid, (B) solid:liquid lipid ratio, (C) total lipid content, and (D) SAA concentration on mean zeta potential (mV).

Figure 3. TEM image of NLC-cinnamon oil formulation.

Figure 4. FTIR band spectra of cinnamon oil, stearic acid, Labrafac, Tween 80, and the chosen NLC-optimized formulation.

Table 5. Comparison of the spreadability and viscosity of pure gel, blank NLC gel, and NLC-cinnamon oil gel (values are expressed as mean ± 1 SD, n = 3).

	Spreadability diameter (cm)	Viscosity (cP) (150 rpm, 22.2 ± 0.5 °C)
Pure gel	2.53 ± 0.59	445.38 ± 28.89
Blank NLC gel	1.82 ± 0.31	689.86 ± 49.47
NLC-cinnamon oil gel	1.46 ± 0.23	977.26 ± 54.24

Figure 5. In vitro release of cinnamon oil from NLC colloid and gel compared to pure cinnamon oil gel in phosphate buffer pH 7.4 containing 30% absolute ethanol at 35 ± 0.5 °C (n = 3).

Table 6. Modeling of drug release from NLC-cinnamon gel, NLC-cinnamon colloid, and cinnamon oil-gel formulations.

	Zero-order	First-order	Higuchi	Korsmeyer–Peppas
	R^2	R^2	R^2	R^2	n
NLC-gel	0.604	0.758	0.953	0.976	0.409
NLC-colloid	–4.000	–1.298	–1.590	0.760	0.141
Cinnamon oil-gel	–0.676	–0.022	0.297	0.794	0.234

Table 7. Comparison of MIC against five strains of P. aeruginosa in formulation and excipients.

	MIC (µg/ml) (n = 5)
Cinnamon oil	2250.00 ± 559.02
Plain gel	N/A
NLC blank gel	N/A
NLC-cinnamon oil colloid	562.00 ± 139.75
NLC-cinnamon oil gel	281.20 ± 69.99

Figure 6. Images of original burn wound (day 0) and after treatment on day 6. Group 1: no treatment, group 2: cinnamon oil, group 3: NLC-cinnamon oil colloid, group 4: NLC blank gel, and group 5: NLC-cinnamon oil gel.

Figure 7. Weight changes during the treatment. Group 1: no treatment, group 2: cinnamon oil, group 3: NLC-cinnamon oil colloid, group 4: NLC blank gel, and group 5: NLC-cinnamon oil gel.

Figure 8. Burn area contraction (cm²) of tested groups. Group 1: no treatment, group 2: cinnamon oil, group 3: NLC-cinnamon oil colloid, group 4: NLC blank gel, and group 5: NLC-cinnamon oil gel (p<.0001).

Figure 9. Rat skin samples after the six-day treatment. (A) The normal skin control group showed intact skin surface epithelium and normal dermal and subcutaneous tissues without inflammation (H&E, ×100). (B) Group 1 – no treatment: wide ulceration of the surface (blue arrow) covered by an excessive amount of fibrinoid material (red arrow). The dermis and the subcutaneous tissue show mixed acute and chronic inflammatory cells (green arrow), H&E ×200. (C) Group 2 – cinnamon oil gel: smaller skin ulcers (blue arrow) covered by fibrinoid material (red arrow). The dermis and subcutaneous tissue show inflammatory infiltrate (green arrow) and fibrosis (black arrow) H&E ×200. (D) Group 3 – NLC-cinnamon oil colloid: wide areas of granulation tissue (green arrow) admixed with fibrosis denoting the healing process (black arrow) H&E ×200. (E) Group 4 – NLC blank gel: focal ulcerated area covered by fibrinoid material (red arrow) with small areas of granulation tissue and fibrosis (black arrow) extending to the dermis and subcutaneous tissue with moderate mixed acute and chronic inflammatory infiltrate (green arrow), H&E ×200. (F) Group 5 – NLC-cinnamon oil gel: wide areas of granulation tissue and fibrosis occupying the whole dermis (black arrow), H&E ×200.