Development of a novel microemulsion for oral absorption enhancement of all-trans retinoic acid

Figures & data

Figure 1 Structure of all-trans retinoic acid.

Table 1 Solubility of ATRA in various surfactants/co-surfactants

Surfactants/co-surfactants	Solubility (mg/mL)
Oleth-5 (Eumulgin 05)	18.88±0.18
Transcutol P	18.53±0.08
PEG-7 glyceryl cocoate (Cetiol HE)	11.46±0.03
Tween 20	11.71±0.01
Tween 80	12.67±0.09
Ethanol	7.44±0.02
Apricot kernel oil PEG-6 esters (Labrafil M 1944CS)	6.11±0.03
Propylene glycol	0.811±0.002

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviation: ATRA, all-trans retinoic acid.

Figure 2 Pseudo-ternary phase diagram composed of fish oil as the oil phase with various surfactant mixtures (A) oleth-5 and Transcutol P (1:1), (B) oleth-5 and Cetiol HE (1:1), (C) oleth-5 and Tween 20 (1:1) and (D) oleth-5 and Tween 80 (1:1) as surfactant and co-surfactant, respectively. The gray area represents the microemulsion region.

Table 2 Formulations, component ratio, ATRA solubility and ATRA loaded in microemulsions

Formulations	Ratio			Solubility of ATRA (mg/mL)	ATRA loaded in microemulsion (mg/mL)
	Oil	Surfactant mixturea	Water
ME 1	10	75	15	11.06±0.02*	10
ME 2	5	80	15	12.45±1.49*	11
ME 3	15	75	10	15.39±0.49*	14
ME 4	5	90	5	20.71±1.84*	18

Notes:

a Surfactant:co-surfactant ratio =1:1 w/w.

* Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 3 Pseudo-ternary phase diagram composed of fish oil as the oil phase with various surfactant and co-surfactant mixture (oleth-5 and Transcutol P) ratios (A) 1:1, (B) 2:1, (C) 3:1 and (D) 4:1. The gray area represents the microemulsion region.

Table 3 Particle size, zeta potential, PDI and electrical conductivity of MEs without ATRA (blank ME) and ATRA-loaded ME

Formulations	Particle size (nm)		Zeta potential (mV)		PDI		Conductivity (µS/cm)
	Blank ME	ATRA loaded ME	Blank ME	ATRA loaded ME	Blank ME	ATRA loaded ME	Blank ME	ATRA loaded ME
ME 1	102.05±38.36	177.9±23.5	0.14±0.13	−0.04±0.21	0.292±0.057	0.28±0.03	75±2	61±1
ME 2	38.1±2.4	42.18±2.65	0.10±0.06	−0.08±0.03	0.35±0.02	0.345±0.05	87±2	70±1
ME 3	92.29±15.69	104.9±34.9	0.087±0.231	−0.0075±0.3704	0.387±0.074	0.318±0.107	51±3	47±5
ME 4	39.81±32.16	167.7±30.1	0.10±0.05	−0.26±0.15	0.313±0.123	0.531±0.034	35±1	38±4

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion; PDI, polydispersity index.

Figure 4 Transmission electron microscopy images of (A) ATRA-loaded ME 1, (B) ATRA-loaded ME 2, (C) ATRA-loaded ME 3 and (D) ATRA-loaded ME 4. Magnification ×200.

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 5 The rheological behaviors of ATRA-loaded ME 1 (Img), ATRA-loaded ME 2 (Img), ATRA-loaded ME 3 (Img) and ATRA-loaded ME 4 (Img). All data represent the (mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Table 4 In vitro release characteristics of ATRA-loaded MEs

Release kinetics	ME 1	ME 2	ME 3	ME 4
Zero order R^2	0.9745±0.0268	0.9428±0.0239	0.9893±0.0075	0.9720±0.0006
First order R^2	0.7329±0.0987	0.8684±0.0225	0.7416±0.0204	0.823±0.081
Higushi R^2	0.8875±0.0921	1.7178±0.4678	0.8817±0.0232	0.8363±0.0018
Zero order release rate (h^−1)	0.5123±0.1647	0.3322±0.0947	0.3141±0.0021	0.2817±0.0409

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 6 The in vitro percent cumulative release profiles of all-trans retinoic acid from the microemulsion formulations ME 1 (Img), ME 2 (Img), ME 3 (Img) and ME 4 (Img). All data represent the mean ± standard deviation (n=3).

Abbreviation: ME, microemulsion.

Table 5 The apparent permeability (P_app) of ATRA from each formulation

Formulations (ATRA concentration, mg/mL)	Papp (×10^−7 cm/s)
Fish oil (1.4)	18.11±1.58
ATRA-loaded ME 1 (10)	16.31±1.41
ATRA-loaded ME 2 (11)	13.73±0.89
ATRA-loaded ME 3 (14)	17.29±4.13
ATRA-loaded ME 4 (18)	24.42±1.77

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 7 Confocal laser scanning microscopy images show x–z plane serial penetration of a porcine intestine treated with (A1) nile red in fish oil and (B1) nile red-loaded ME 4 at a time of 3 h, scale bar represents 100 µm. The (A2) and (B2) images are the intensity projections through the z-axis of (A1) and (B1), respectively. The scale bar represents 100 µm.

Abbreviation: ME, microemulsion.

Figure 8 Comparison of fluorescence intensity profiles of nile red at various depths into the intestine shown in Figure 7. All data represent the mean ± standard deviation.

Abbreviation: AU, arbitrary unit.

Table 6 The percent cytotoxicity of blank fish oil, blank ME 4 ATRA in fish oil and ATRA-loaded ME 4

Formulations	% cytotoxicity
Blank fish oil	15.18±0.12
Blank ME 4	5.61±6.23
ATRA in fish oil	17.18±7.68
ATRA-loaded ME 4	13.91±2.13

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 9 H&E-stained porcine intestine treated with (A) phosphate-buffered saline, (B) 3% w/w Triton X-100, (C) ATRA in fish oil and (D) ATRA-loaded ME 4.

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 10 The percentage of ATRA remaining in ME 4 after storage for 0, 1, 2, 3, 4, 5 and 6 months after preparation at 25°C (■) and 40°C (□). Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Table 7 The mean particle size of ATRA-loaded ME 4 at baseline, 1, 2, 3, 4, 5 and 6 months after preparation stored at 25°C and 40°C

Temperature (°C)	Size (nm)
	Baseline	1 month	2 months	3 months	4 months	5 months	6 months
25	167.7±30.1	161.43±34.67	174.53±35.45	163.17±38.29	162.53±68.26	160.77±36.03	152.67±27.96
40	167.7±30.1	185.8±35.9	212.27±36.24	160.13±31.58	219.6±79.8	199.7±42.3	181.01±13.86

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Table 8 Zeta potential of ATRA-loaded ME 4 at baseline, 1, 2, 3, 4, 5 and 6 months after preparation stored at 25°C and 40°C

Temperature (°C)	Zeta potential (mV)
	Baseline	1 month	2 months	3 months	4 months	5 months	6 months
25	−0.26±0.15	−0.21±0.05	−0.24±0.08	−0.26±0.17	−0.22±0.11	−0.28±0.03	−0.37±0.06
40	−0.26±0.15	−0.23±0.06	−0.31± 0.06	−0.26±0.07	−0.31±0.27	−0.25±0.08	−0.22±0.08

Note: Each value represents the mean ± standard deviation (n=3).

Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.