Spironolactone hyaluronic acid enriched cerosomes (HAECs) for topical management of hirsutism: in silico studies, statistical optimization, ex vivo, and in vivo studies

Figures & data

Table 1. Atomic composition of the investigated SP-PC-HA simulated systems.

Solvation State	composition (Number of molecules)
	SP	PC	HA	Chloroform	Ethanol	Water	Total
100% Water	1	1	1	–	–	4053	4056
100% Chloroform	1	1	1	704	–	–	707
Chloroform + ethanol	1	1	1	352	635	–	990

Abbreviation: SP, Spironolactone; PC, phospholipid, and HA, hyaluronic acid.

Table 2. D-optimal design used for optimization of SP-loaded HAECs.

Factors (independent variables)	Factor type	Levels
		(−1)	(+1)
X 1: Ceramide amount (mg)	Numeric	5	15
X 2: HA (mg)	Numeric	5	15
X 3: EA type	Categoric	Kolliphor RH40	Kolliphor EL
Responses (dependent variables)	Desirability constraints
Y 1: EE%	Maximize
Y 2: PS (nm)	Minimize
Y 3: PDI	Minimize

Abbreviation: HA: hyaluronic acid; EA: edge activator; EE%: entrapment efficiency percent; SP: Spironolactone; PS: particle size; PDI: polydispersity index and HAECs; hyaluronic acid enriched cerosomes.

Figure 1. Binding poses of the spironolactone phosphatidyl choline docked complex. (a) In absence of the hyaluronic acid monomer. (b) In combination with the hyaluronic acid monomer (magenta sticks) serving as an anionic formulation additive. Predicted binding interactions are illustrated between the drug molecule; spironolactone (yellow sticks) and of the hyaluronic acid monomer; phosphatidyl choline (green sticks). More favored complex stability was assigned for the spironolactone–phosphatidyl choline in combination with hyaluronic acid. Polar interactions (hydrogen bonding), discussed within the text, are depicted as black dashed lines.

Figure 2. Conformation alterations time evolution of SPH ternary complex throughout the explicit molecular dynamics simulation at different solvation system. The thermodynamic movements and stability of formulation components; spironolactone (yellow sticks), hyaluronic acid monomer (magenta sticks), and phosphatidyl choline (green sticks) were monitored over MD simulation trajectories within (a) 100% chloroform; (b) combined chloroform/alcohol; (c) 100% water solvated systems at captured at different snapshots (1) 0.2 ns; (2) 0.4 ns; (3) 0.6 ns; (4) 0.8 ns; and (5) 1 ns. Polar interactions (hydrogen bonding) are depicted as black dashed lines. (d) Molecular surface 3 D representation of the cone micellar configuration of optimized SPH in chloroform: alcohol (1:1) combination (left panel), as well as the inverted cone micellar configuration of 100% water (right panel). Molecular surface representations were illustrated in colors being previously assigned for the optimized SPH components; yellow, magenta, and green are for spironolactone, hyaluronic acid monomer, and phosphatidyl choline, respectively.

Table 3. Experimental runs, independent variables and measured responses of SP- HAECs.

Formula^a	Composition			Y 1: EE%	Y 2: PS (nm)	Y 3: PDI
	X 1:Ceramide amount (mg)	X 2: HA (mg)	X 3: EA type
HAEC 1	15	5	Kolliphor RH40	84.65 ± 1.36	329.61 ± 1.55	0.458 ± 0.009
HAEC 2	10	10	Kolliphor RH40	75.09 ± 2.08	300.00 ± 7.00	0.601 ± 0.031
HAEC 3	10	5	Kolliphor RH40	68.82 ± 1.19	319.00 ± 74.03	0.643 ± 0.094
HAEC 4	5	5	Kolliphor RH40	71.59 ± 5.13	350.00 ± 20.00	0.462 ± 0.057
HAEC 5	15	15	Kolliphor RH40	93.54 ± 2.06	297.30 ± 8.62	0.639 ± 0.049
HAEC 6	15	15	Kolliphor RH40	96.76 ± 1.30	292.60 ± 10.54	0.646 ± 0.042
HAEC 7	5	15	Kolliphor RH40	83.40 ± 1.20	297.30 ± 5.05	0.474 ± 0.025
HAEC 8	5	15	Kolliphor RH40	81.18 ± 1.96	292.60 ± 6.08	0.471 ± 0.020
HAEC 9	5	5	Kolliphor EL	44.78 ± 1.02	258.80 ± 2.86	0.489 ± 0.007
HAEC 10	5	5	Kolliphor EL	43.55 ± 1.45	261.8 ± 1.90	0.481 ± 0.005
HAEC 11	10	7.5	Kolliphor EL	68.23 ± 14.75	320.00 ± 4.00	0.455 ± 0.019
HAEC 12	10	15	Kolliphor EL	75.89 ± 16.38	340.10 ± 1.62	0.762 ± 0.023
HAEC 13	5	15	Kolliphor EL	68.76 ± 1.07	292.40 ± 14.50	0.587 ± 0.110
HAEC 14	15	15	Kolliphor EL	70.51 ± 0.89	459.33 ± 18.87	0.652 ± 0.077
HAEC 15	15	5	Kolliphor EL	70.29 ± 7.88	400.50 ± 20.50	0.612 ± 0.043

Presented values are the mean ± SD (n = 3).

^aAll formulae contained 10 mg SP and 100 mg phosphatidylcholine, in a volume of 10 mL.

Abbreviation: HA, hyaluronic acid; EA, edge activator; EE%, entrapment efficiency percent; PS, particle size; PDI, polydispersity index; SP, spironolactone; HAECs, hyaluronic acid enriched cerosomes.

Table 4. Output data of the D-optimal design analysis of SP-HAECs.

Response	Y 1: EE% (%)	Y 2: PS (nm)
Minimum	43.6	258.8
Maximum	96.8	459.3
Ratio	2.22	1.77
Model	Linear	Quadratic
R-squared	0.9238	0.9970
Adjusted R-squared	0.9030	0.9930
Predicted R-squared	0.8545	0.9705
Adequate precision	19.643	57.726
Significant terms (coded factors)^a	A, B, and C	A, C, AB, AC, BC, A^2
Regression equation (in terms of actual factors)	EA type Kolliphor RH40 EE% = +50.92217 +1.46752 × Ceramide amount(mg) +1.50436 × HA amount(mg)	EA type Kolliphor RH40 PS(nm) = +458.60178 −26.42834 × Ceramide amount(mg) +0.48737 × HA amount(mg) +0.24540 × Ceramide amount(mg) × Hyaluronic acid amount(mg) +1.14427 × Ceramide amount(mg)^2 −0.38439 × HA amount(mg)^2
	EA type Kolliphor EL EE% = +34.86665 +1.46752 × Ceramide amount(mg) +1.50436 × HA amount(mg)	EA type Kolliphor EL PS(nm) = +238.93537 −10.09234 × Ceramide amount(mg) +9.48687 × HA amount(mg) +0.24540 × Ceramide amount(mg) × Hyaluronic acid amount(mg) +1.14427 × Ceramide amount(mg)^2 −0.38439 × HA amount(mg)^2

^aA, Ceramide amount; B, HA amount; C, EA type.

Abbreviation: HA, hyaluronic acid; EA, edge activator; EE%, entrapment efficiency percent; PS, particle size; HAECs, hyaluronic acid enriched cerosomes.

Figure 3. Effect of formulation variables on EE% of SP-HAECs (a–c), PS (d–f), while (g–i) show the effect of significant interactions on PS. Abbreviations: EE%, Entrapment efficiency percentage; PS, Particle size; SP, spironolactone; HAECs, Hyaluronic acid enriched cerosomes.

Table 5. Effect of storage on different measurements of OHAEC.

Parameter^a	Fresh OHAEC	Stored OHAEC (3 months)
EE%	89.3 ± 0.3	88.5 ± 1.6
PS (nm)	261.8 ± 7.0	267.8 ± 10.1
ZP (mV)	−9.0 ± 1.1	−7.3 ± 2.4
PDI	0.482 ± 0.07	0.465 ± 0.06

^aMean ± SD (n = 3).

Abbreviation: EE%, entrapment efficiency percent; PS, particle size; PDI, polydispersity index; ZP, zeta potential; OHAECs, optimal hyaluronic acid enriched cerosomes.

Figure 4. Transmission electron micrographs of SP OHAEC showing mixed vesicular and tubular appearances. Abbreviation: OHAEC, optimal hyaluronic acid enriched cerosomes.

Figure 5. Ex vivoprofile of SP from OHAEC, compared to its aqueous suspension. Abbreviation: SP: spironolactone, OHAEC: optimal hyaluronic acid enriched cerosomes.

Table 6. Ex vivo permeation and deposition parameters of OHAEC, compared to SP suspension.

Parameter^a	OHAEC	SP-suspension
Q24 (µg/cm^2)	0.43 ± 0.08	6.07 ± 0.26
Dep24 (µg/cm^2)	55.45 ± 0.35	42.74 ± 5.82
J max (µg/h cm^2)	0.018 ± 0.003	0.253 ± 0.011
LAEI	129.78 ± 11.22	7.02 ± 0.66
LAEI ratio	18.48	–

^aMean ± SD (n = 3).

Abbreviation: Q₂₄, cumulative amount permeated per unit area after 24 h; Dep₂₄, cumulative amount deposited per unit area after 24 h; J _max, maximum average flux after 24 h; LAEI, Local accumulation efficiency index; SP, spironolactone; OHAECs, optimal hyaluronic acid enriched cerosomes.

Figure 6. Light microscope photomicrographs showing histopathological sections (hematoxylin and eosin stained) of rat skin normal control (group I), rat skin treated with SP suspension (group II) and rat skin treated with OHAEC (group III) with magnification power of 16X to illustrate all skin layers (Left side) and magnification power of 40X to identify the epidermis and dermis (Right side). Abbreviation: SP: spironolactone, OHAEC: optimal hyaluronic acid enriched cerosomes.

Figure 7. In vivo skin deposition profile of SP from OHAEC, compared to its aqueous suspension. Abbreviation: SP: spironolactone, OHAEC: optimal hyaluronic acid enriched cerosomes.