Accelerated infected wound healing by topical application of encapsulated Rosemary essential oil into nanostructured lipid carriers

Figures & data

Figure 1. (A) Scanning electron microscopy (SEM) image of optimum REO-NLCs formulation. The profile of (B) particle size and (C) zeta potential for REO-NLCs formulation.

Table 1. The mean of diameter zone (mm) in experimental groups.

Groups	E. coli	S. typhimurium	P. aeruginosa	S. aureus	S. epidermidis	B. anthracis	S. pneumonia	L. monocytogenes
REO	11.43 ± 1.55^A,d	7.38 ± 3.47^B,c	12.05 ± 2.36^A,c	12.01 ± 1.59^A,c	11.98 ± 1.95^A,c	0.00 ± 0.00^C,d	0.00 ± 0.00^C,e	10.99 ± 2.13^A,d
REO- NLCs	12.98 ± 2.71^A,d	6.99 ± 4.93^BC,c	14.09 ± 2.07^A,c	10.07 ± 3.61^B,c	13.83 ± 273^A,c	0.00 ± 0.00^D,d	5.75 ± 1.04^C,d	12.73 ± 1.99^A,d
Gentamicin	21.70 ± 0.77^c	20.45 ± 0.67^b	35.19 ± 0.39^b	37.30 ± 0.02^b	35.39 ± 0.21^b	14.11 ± 0.16^c	27.53 ± 0.53^c	27.65 ± 0.47^c
Penicillin	40.81 ± 0.12^a	27.62 ± 0.59^a	46.78 ± 0.57^a	44.26 ± 0.34^a	42.28 ± 0.58^a	31.21 ± 1.99^a	39.32 ± 0.42^a	37.40 ± 0.56^a
Mupirocin	35.73 ± 0.41^b	26.36 ± 0.52^ab	42.14 ± 0.50^a	41.12 ± 0.39^a	41.41 ± 0.92^a	30.56 ± 0.71^b	36.41 ± 0.15^b	31.92 ± 0.61^a
Blank	0.00 ± 0.00^e	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^e	0.00 ± 0.00^d
DMSO	0.00 ± 0.00^e	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.s00^e	0.00 ± 0.00^d
P-value	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001

Superscripts (a–e) show significant differences among each column. Superscripts (A–D) show significant differences in each row in REO and REO-loaded NLCs. The data are shown as mean ± SD.

Table 2. The data for MIC and MBC in REO and REO-NLCs groups.

	MIC		MBC
Groups	REO	REO-loaded NLCs	REO	REO-loaded NLCs
E. coli	6.53 ± 1.49^d	8.92 ± 1.75^c	9.71 ± 1.59^cd	11.73 ± 1.71^c
S. typhimurium	18.57 ± 0.78^b	15.75 ± 1.28^b	17.99 ± 0.72^b	17.59 ± 1.31^b
P. aeruginosa	4.99 ± 1.59^c	5.34 ± 1.27^d	7.91 ± 1.91^d	7.21 ± 1.81^d
S. aureus	6.71 ± 1.41^d	10.13 ± 1.67^c	10.11 ± 1.61^c	11.61 ± 1.81^c
S. epidermidis	5.21 ± 1.51^b	7.93 ± 1.84^ab	7.01 ± 1.62^d	10.27 ± 1.87^c
B. anthracis	19.50 ± 0.53^a	20.70 ± 0.41^a	19.39 ± 0.14^a	20.00 ± 0.00^a
S. pneumonia	20.00 ± 0.00^a	17.89 ± 0.19^a	19.71 ± 0.23^a	20.00 ± 0.00^a
L. monocytogenes	9.43 ± 1.78^c	9.35 ± 1.71^c	12.51 ± 1.91^c	12.69 ± 1.75^c
P-value	0.001	0.001	0.001	0.001

Superscripts (a–d) show significant differences in each column. The data are shown as mean ± SD.

Figure 2. Effect of REO and REO-NLCs gels on wound area (mm²) on different days. Six animals in each group. Superscripts (a–b) show significant differences among groups on same day.

Figure 3. Effect of REO and REO-NLCs gels on total bacterial count (CFU/g) in different days. Six animals in each group. Superscripts (a–b) show significant differences between other groups and control.

Figure 4. Histological section from wound area on day 3 after wound induction; immuno-fluorescent staining for fibroblast are presented in first row (A1–D1). See that REO-NLCs and the REO significantly stimulated the fibroblasts infiltration. Figure A2–D2 are representing the granulation tissue generation in different groups. See well-formed granulation tissue in REO and REO-NLCs-treated groups versus mupirocin and control animals. The software analyses for fibroblast distribution are represented in Figure A3-D3. See intensive fibroblast distribution in REO-NLCs-treated group (A: Control, B: Mupirocin-treated, C: REO-treated and D: REO-NLCs-treated).

Figure 5. Histological section from wound area on day 14 after wound induction; (A1–D1) immuno-fluorescent staining for fibroblast are presented in first row (A1–D1). See that REO-NLCs and the REO significantly stimulated the fibroblasts infiltration. Figure A2–D2 are representing Masson-trichrome staining techniques for collagen deposition in dermis (DS). See the well-formed re-epithelialization in REO and REO-NLCs-treated groups compared with mupirocin-treated and control animals. See hair follicle sprouting (HF) and sebaceous glands (SG) in REO and REO-NLCs-treated cross sections. The software analyses for fibroblast distribution are represented in Figure A3–D3. See intensive fibroblast distribution in REO-NLCs-treated group (A: Control, B: Mupirocin-treated, C: REO-treated and D: REO-NLCs-treated).

Table 3. Effect of REO and REO-NLCs gels on histological scorings on different days.

Groups	Edema	Vascularization	Fibroblast disturbation	Granulation tissue	Collagen synthesis	Epithelization
Day 3
Control	++++	–	+	+	–	–
Mupirocin	+++	+	+	+	+	–
REO	+++	+	+	+	+	–
PEO-NLCs	++	++	++	+	+	–
Day 7
Control	+++	+	+	+	+	+
Mupirocin	++	++	++	++	++	++
REO	++	++	++	++	++	++
REO- NLCs	+	+++	+++	+++	+++	+++
Day 14
Control	++	++	++	++	++	++
Mupirocin	–	+++	+++	+++	+++	+++
REO	–	+++	+++	+++	+++	+++
REO-NLCs	–	++++	++++	++++	++++	++++

Figure 6. Effect of REO and REO-NLCs gels on IL-3 (A) IL-10 (B), VEGF (C) and SDF-1α (D) on different days. Six animals in each group. Superscripts (a–b) show significant differences between other groups and control sham.