Microemulsions and Nanoemulsions for Topical Delivery of Tripeptide-3: From Design of Experiment to Anti-Sebum Efficacy on Facial Skin

Abstract

The targeted delivery of a hydrophilic Tripeptide-3 to the skin using microemulsions or nanoemulsions for facial oil reduction was the focus of this study. The impact factors affecting oil/water transparent dispersion formation, such as the surfactant system, HLB value, and co-solvent, were identified through the water titration method and pseudoternary phase diagram plots. The interfacial tension between caprylic/capric triglyceride (CCT oil) and water was significantly reduced by the surfactant/co-surfactant combination (S_mix) of Cremophore^® RH40 and a double-tails co-surfactant, polyglycerol-3-diisostearate, at an HLB of 13 together with a water-to-co-solvent (PG) ratio of 1:1. A two-level full factorial design of experiment (FFD-DoE) emphasized the independent variables of the HLB value, co-solvent, and CCT oil contents affecting the optimal compositions for micro- or nanoemulsion formation. The low-energy spontaneous emulsification of the optimized combination at a low S_mix content (10%) yielded the translucent oil-in-water Tripeptide-3 nanoemulsions with an internal droplet size of 25.7 ± 1.20 nm, a narrow polydispersity index of 0.237 ± 0.129, and 70.6 ± 0.58% transmittance. The in vitro skin permeation study revealed a significantly higher skin penetration and retention of the Tripeptide-3 nanoemulsions compared to the high surfactant microemulsions and coarse emulsions. Skin irritation and oil control efficacy were evaluated in healthy volunteers before and after product application for 28 days. The obtained nanoemulsions not only decreased sebum production but also enhanced skin moisture levels. In conclusion, the meticulously designed nanoemulsions, incorporating suitable excipients, show a promising delivery system for hydrophilic peptides to control sebum overproduction in oily facial skin.

Keywords: Tripeptide-3; anti-sebum; dermal delivery; design of experiment; efficacy; microemulsions; nanoemulsions; oil-control; topical.

Figure 1

Illustration of Tripeptide-3, surfactant, and co-solvent structures used in the study.

Figure 2

Pseudoternary phase diagrams and the microemulsified area (gray) of various CCT/water/S_mix as a function of the HLB 11 and 13. Area (%) of microemulsion formation of the Cremophor^® RH40 and Span^® 20 at HLB 11 (a) and HLB 13 (b) were compared to the area from the Cremophor^® RH40 and Span^® 80 at HLB 11 (c) and HLB 13 (d), respectively. The S_mix of Cremophor^® RH40 and Span^® 20 at HLB 13 were selected for further evaluation for the effect of co-solvent due to the highest microemulsion area was formed.

Figure 3

Pseudoternary phase diagrams and the microemulsified area of various CCT/S_mix Cremophor^® RH40 and Span^® 20 at HLB 13 as a function of an aqueous phase. Area (%) of microemulsion formation of the water-to-co-solvent ratios of 1:1, 2:1, and 3:1 were compared between two co-solvents, propylene glycol (PG) (a–c) and ethanol (EtOH) (d–f). Propylene glycol with a 1:1 water ratio (a) was selected for further study to assess the effect of double-tailed co-surfactants. This choice was made due to the high microemulsion area obtained and its superior skin compatibility compared to ethanol (EtOH).

Figure 4

The effect Lameform^® TGI as a co-surfactant: (a) pseudoternary phase diagram and the microemulsified area of CCT/S_mix Cremophor^® RH40 and Lameform^® TGI at HLB 13 as a function of an aqueous phase (water:PG, 1:1); (b) MEs from point B, C, D, and the optimized formulation derived from point A; (c) polarized light microscope images show isotropic microstructure of all formulations. In the pseudoternary phase diagram (a), point A was selected for fine-tuned optimization using the design of experiment, while points B, C, and D were selected as the conventional MEs for comparison of skin permeability.

Figure 5

The Pareto chart obtained from the Design Expert^® program demonstrates a statistically significant variable factor (above t-value limit) impact on responses: (a) indicates X₃ (oil content) and interaction between X₁ (HLB value) and X₂ (PG content) exhibited statistically significant impact on particle sizes of ME; (b) shows statistically significant effect of oil content on percent transmittance of ME; and (c) demonstrates X₁ (HLB value) and X₂ (PG content) interaction effect on particle sizes of ME from DoE experiment when oil content was constant; the lowest particle size (■ black line) was formed when HLB value of S_mix and % PG were 13% and 20% in the formulation.

Figure 6

Stress stability studies of micro- and nanoemulsion preparations from the DoE study (a) overnight after preparation; (b) 6 cycles of heating and cooling between 4 and 40 °C, each for a period of 48 h, and (c) after 30 min of 10,000 rpm (25,830× g) centrifugation.

Figure 7

Physical and chemical stability of the optimized formulation with adjusted pH of 4.5, 5.0, and 6.0 at Day 0 (a). After six heating/cooling cycles, pH 5.0 shows slight yellow (B), while pH 6.0 turns to full yellow (C). No color change occurred in pH 4.5 formulation (A) (b). The HPLC chromatograms of Tripeptide-3 revealed the chemical stability of the Tripeptide-3 in the optimized formulation at different pH: (c) represents Day 0, (d) represents after 6 H/C cycle of optimized formulations, with (A) in pH 4.5, (B) in pH 5.0, (C) in pH 6.0, and (D) a standard Tripeptide-3. Paracetamol, an internal standard, and the Tripeptide-3 peaks were eluted at retention times of 7.6 min and 20.5 min., respectively.

Figure 8

Comparative amounts of Tripeptide-3 in different formulations across the membrane and accumulated in the receivers from Franz diffusion study (a), and skin flux determined from each formulation showing statistically significant higher percutaneous absorption rate of Tripeptide-3 optimized nanoemulsion formulation compared with the MEs and emulsion (b); Tripeptide-3 amount per cm² membrane retained in the skin from each formulation after 12 h of the permeation study (c). Each value represents the mean ± SD (n = 3). *** p < 0.001; **** p < 0.0001.

Figure 9

Dermal irritancy and efficacy of Tripeptide-3 nanoemulsion from clinical evaluation: (a) skin irritation comparison (PII values) from Tripeptide NE, positive and negative controls, (b) skin moisture improvement when applied on facial zones, and (c) reduction of skin oiliness comparison between different facial zones on day 14 and day 28. Mean ± SD, n = 23 (paired t-test, p < 0.05, on day 0 vs. day 14 and day 18).