Table 2.

Published approaches of Stratum Corneum surrogates (SCS) incorporating SC lipid biomimetic models for permeation studies.

SC Lipid Model Composition	Porous Substrate	Characterisation Techniques	Model Compounds	Main Outcomes	Ref.
Airbrush approaches
Cer:Chol:FFA (1:1:1 mol ratio) Cer = [EOS]:[NS]:[NP24]: [AS]:[NP16]:[AP] (15:51:16:4:9:5 molar ratio) FFA = PA:SA:AA:BA:TA:LA:CA (1.3:3.3:6.7:41.7:5.4:36.8:4.7 molar ratio)	Polycarbonate 50 nm filter	SWAXS and CryoEM	n.a.	Airbrush is able to homogeneously spray a SC mixture onto a polycarbonate filter. Synthetic lipids in the SCS resembled the orientation of the ILM of SC: the width of the arcs of SCS were close to the ones in the SC. SCS characteristics showed great potential for use as a skin barrier diffusion model.	[58]
		Diffusion studies	PABA, ethyl-PABA, and butyl-PABA	Barrier properties of SCS prepared with a 12-μm-thick lipid layer are similar to those of isolated human SC. SCS model can be easily tuned by adjusting the lipid mixture to mimick different healthy and diseased skin.	[30]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS]:[NP24]: [AS]:[NP16]:[AP] (15:51:16:4:9:5 molar ratio) FFA = PA:SA:AA:BA:TA:LA:CA (1.8:4:7.7:42.6:5.2:34.7:4.1 molar ratio)	Polycarbonate 50 nm filter	Different airbrush methods, SAXS, and diffusion studies	Benzoic acid	Irrespective of the preparation method, benzoic acid permeation profiles were very similar to human SC. While the rotor method is claimed to increase efficiency and reproducibility, the Linomat method reduced the lipid loss during lipid airbrush and was thus selected as the best method to spray lipids on porous substrate.	[79]
Cer:Chol:FFA:ChSO4 (Variable ratio) Cer = [EOS]:[NS]:[NP24]: [AS]:[NP16]:[AP] (15:51:16:4:9:5 molar ratio) FFA = PA:SA:AA:BA:TA:LA:CA (1.8:4:7.7:42.6:5.2:34.7:4.1 mol%)	Polycarbonate 50 nm filter	FTIR, SAXS, and permeability studies	Benzoic acid	Two SCS models showed significant changes in the benzoic acid steady-state flux: an excess of Chol decreased flux, while an excess of ChSO4 increased the flux. Lipid composition disorders in psoriasis and dry skin may not be responsible for impaired skin barrier function in vivo.	[171]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS]:[NP]:[AP] (15:51:16:4:9:5 molar ratio) FFA = PA:SA:AA:BA:TA:LA:CA or MyA:PA:SA:AA:BA (Variable ratio)	Polycarbonate 50 nm filter	SAXS, EM, and permeation studies	Benzoic acid	Permeability of the SC system to benzoic acid closely follows that of human SC at temperatures ranging from 31 to 43 °C.	[172]
Cer[AP]:Chol:PA:ChS (55:25:15:5 wt%)	Polycarbonate 50 nm filter	SAXS, polarisation, confocal and ESEM microscopies, Raman imaging, FTIR, and diffusion studies	Urea	A continuous uniformly distributed lipid layer on polycarbonate filters was obtained. Membranes showed higher barrier function than isolated human SC.	[173]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS]:[NP24]:[NP16] (16:56:18:10 mol%)	Polycarbonate 50 nm filter	FTIR and interaction studies	γ-undecalactone, DDA, and diethyl 1,4-cyclohexanedicarboxylate	The dose-dependent effects of the chemicals on the lateral molecular packing in the SCS were qualitatively identical to those observed by IR spectroscopy in human SC.	[174]
Cer[NS]:Chol:PA (1:1:1 molar ratio)	Polycarbonate 50 nm filter	Laurdan fluorescence, SEM, Raman scattering, and permeability studies	Benzoic acid and CAF	SCS showed continuous coverage of gel-phase lipids, allowing it to be used in permeability studies. Barrier properties in the same order as ‘real’ SC.	[175]
Cer:Chol:FFA (1:1:1 molar ratio) pigCer or synCer = [EOS]:[EOP]:[NS]:[NP24]: [AS]:[NP16]:[AP] (Variable ratio) FFA = PA:SA:AA:BA:TA:LA:CA (1.8:4:7.7:42.6:5.2:34.7:4.1 mol%)	Polycarbonate 50 nm filter	SAXS, FTIR, and permeation studies	HC	Reduced barrier in SCS prepared with pigCers compared to synthetic Cers. Chain length distribution affects the lipid barrier.	[164]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS]:[NP24]: [AS]:[NP16]:[AP] (15:51:16:4:9:5 molar ratio) FFA = PA:SA:AA:BA:TA:LA:CA (1.8:4:7.7:42.6:5.2:34.7:4.1 mol%)	Polycarbonate 50 nm filter	SAXS, FTIR, TEWL, and permeation studies	HC	Presence of MUFAs in the FFA fraction enhanced SCS permeability by inducing the formation of hexagonal lateral packing. Lipid barrier function decreased with the increased unsaturation degree.	[176]
aCer:Chol:LA:ChS (1:1:1 molar ratio + 5 wt%)	Polycarbonate 15 nm filter	IR, SWAXS, and permeation studies	TH and IND	Strong permeabilisation effects of pentadecasphingosine Cer with short acyls. SCS permeability to relatively small and hydrophilic compounds was more sensitive to lamellar assembly than to the overall lipid chain order and packing.	[177]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [NS] or [NS]:[NP24]:[AS]:[NP16]: [AP] (60:19:5:11:6 mol%) FFA = PA:SA:AA:BA:LA(1.8:4:7.6:47.8:38.9 mol%)	Polycarbonate 50 nm filter	FTIR, XRD, and permeability studies	ethyl-PABA	Ethyl-PABA flux was affected by the SCS FFA composition: flux was higher when a mixture of FFA was used than when a single FFA C24 was present. Ethyl-PABA flux was not affected by the Cer composition.	[178]
Cer:Chol:SA:ChS (1:1:1 molar ratio + 5 wt%) Cer = [NP]:[AP] (Variable ratio)	Polycarbonate 15 nm filter	IR, EI, EM, and permeation studies	TH and IND	SCS based on one Cer showed greater permeability for both chemicals (Cer[AP] more than Cer[NP]) than those based on their mixture, without significant difference from the flux on porcine skin.	[179]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS], [EOS]:[AS], [EOS]:[NP] or [EOS]:[AP] (40:60 mol%) FFA = PA:SA:AA:BA:LA (1.8:4:7.6:47.8:38.8 mol%)	Polycarbonate 50 nm filter	FTIR, X-ray, and permeability studies	ethyl-PABA	Due to distinct in-phase behaviour, lipid chain packing, and headgroup interactions, sphingosine-based Cer SCS resulted in a higher permeability than the phytosphingosine SCS.	[169]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS]:[NS24]: [NS16]:[NP]:[AS24]: [AS16]:[AP24] (Variable ratio) FFA = PA:SA:AA:BA:LA (Variable ratio)	Polycarbonate 50 nm filter	FTIR, SWAXS, permeability studies, and TEWL	ethyl-PABA	Changes in the SCS lipid compositions mimicking atopic dermatitis patients resulted in decreased barrier capability; FFA compositional changes showed a greater impact on barrier function than changes in the Cer profile.	[180]
hCer:Chol:FFA:ChS (1:1:1 molar ratio + 5 wt%) FFA = PA:SA:AA:BA:LA (1.8:4:7.6:47.8:38.8 mol%) Cer[NS]:Cer[EOS]:Chol: LA:ChS (Variable ratio)	Polycarbonate 15 nm filter	WAXS, FTIR, TEWL, electrical impedance, and permeation studies	TH and IND	While 40% of the Chol SCS was less permeable than the control, Chol depletion did not compromise the barrier function to water, TH, and IND.	[181]
aCer[NS]:Chol:LA:ChS (1:1:1 molar ratio + 5 wt%)	Polycarbonate 15 nm filter	XRD, IR spectroscopy, permeability studies, water loss, and electrical impedance	TH and IND	SCS containing 3-deoxy-Cer, as well as 1-deoxy-Cer, had similar permeabilities. Methylation of the Cer[NS] nitrogen improved its miscibility with Chol, disordered lipid chains, and increased the permeability compared to the physiological Cer[NS] control.	[36]
Cer:Chol:LA (1:1:1 molar ratio) Cer = [EOS]:[NS24] or [EOS:NS16] (Variable ratio)	Polycarbonate 50 nm filter	FTIR, SAXS, and permeation studies	ethyl-PABA	Ethyl-PABA permeability increased significantly when ≥50% of Cer[NS24] was substituted with Cer[NS16].	[182]
Cer:Chol:FFA+ChS (1:1:1 molar ratio + 5 wt%) Cer = [NS], R-/S-[AS], [NdS], R-/S-[AdS], [NP] or R-/S-[AP]	Polycarbonate 15 nm filters	XRD, FTIR, and permeability studies	TH and IND	Mixed effects highly dependent on the sphingoid base chain, configuration, and permeability marker used.	[37]
Cer:Chol:FFA (1:1:1 molar ratio) Cer = [EOS]:[NS] (10–90:90–10 mol%) FFA = PA:SA:AA:BA:LA (1.8:4:7.6:47.8:38.8 mol%)	Polycarbonate 50 nm filters	SAXS, FTIR, permeation studies, and TEWL	ethyl-PABA	Steady-state flux only increased when the Cer[EOS] content was raised to 50%, becoming significantly higher when it was raised to ≥70%.	[183]
Cer:Chol:FFA:ChS (1:1:1 molar ratio + 5 wt%) Cer = [EOP]:[EOdS]:[EOS]: [AP]:[AS]:[NP]:[NS]: [NdS]:[AdS] (Variable ratio) FFA = BA:LA:AA:SA:PA (47.1:41.4:6.9:3.3:1.3 mol%)	Polycarbonate 15 nm filter	XRD, FTIR, electrical impedance, and permeability studies	TH and IND	SCS with 10–20 mol% of acylCers mimick the nanostructure and permeability of healthy skin barrier lipids, while 0–5 mol% mimick some hallmarks of a diseased skin lipid barrier.	[184]
				Permeability studies indicated that ω-OHCers could not be substituted by ω-O-acylCers. Absence of ω-O-acylCers resulted in diminished barrier properties.	[185]
Cer[NS]:Chol:FFA:ChS (Variable ratio) FFA = PA:SA:AA:BA:LA (1.8:3.9:7.5:47.8:39 mol%)	Polycarbonate 15 nm filters	H-NMR, SAXS, WAXS, zeta potential, and permeability studies	TH and IND	High ChS:Chol ratio increased the SCS permeability.	[186]
Cer[NS]:Chol:PA (1:1:1 molar ratio)	Polycarbonate 50 nm filters	SEM, DLS, and permeation studies	CAF	CAF showed similar and comparable permeability profiles in SCS using two different approaches and in pig skin.	[187]
Skin-PAMPATM approaches
Certramides:Chol:SA (0 to 1:1:1 molar ratio)	Multiscreen-IP 450 nm pores	Permeability studies	CIP, NFD, and VER	A relationship between the SCS concentration of certramides (from 0 to 100%) and the permeability of the compounds was found. Strongest effect on permeability was caused by the certramide C12–C16.	[80]
Certramides:Chol:SA: Si oil	StirwellTM PAMPA	Permeability studies	APAP, DCF, FUR, NAP, PEF, TH, and VER	By comparison to three different databases, skin–PAMPA demonstrated high prediction capability.	[188]
PVPA-based approaches
E80:BSCCer:Chol:PA:ChS (77:23:0:0:0 or 50:27.5:12.5:7.5:2.5 wt%)	Mixed cellulose ester 650 nm filters	Permeation studies	Flu, IBP, IND, SAL, FITC-dextran, and CAL	PVPA approaches were able to distinguish between substances with different degrees of transdermal absorption.	[189]
E80:BSCCer:Chol:PA:ChS (50:27.5:12.5:2.5:7.5 wt%)	Mixed cellulose ester 650 nm filters	Permeation and stability studies	Liposomal formulations of DCF	Adaptations to the original systems were performed towards nanoformulation testing. All liposomal formulations of DCF exhibited significantly higher permeabilities than DCF solution. PVPA has shown to be valuable for screening and the optimisation of lipid nanoformulations at the preformulation stage.	[190]
E80:BSCCer:Chol:PA:ChS (77:23:0:0:0 or 50:27.5:12.5:7.5:2.5 wt%)	Mixed cellulose ester 650 nm filters	Stability and permeation studies	CAF, ACV, CPL, and CAL (liposomal formulation and/or aqueous solution)	PVPA systems were more promising in detecting permeability differences between aqueous solution and liposomal formulations than EpiSkin®. PVPA showed greater stability than EpiSkin® (up to 2 weeks against 3-day testing window).	[191]
		Diffusion studies	CAF, DCF, CAP and CAL	SCS showed good correlation by ranking model drugs similarly to the ranks obtained using a pig ear skin model and were comparable to the literature on permeation through healthy human skin.	[192]
EPC/SPC:BSCCer:Chol:PA:ChS (25–80:41–11:18–5: 3.75–1:11.25–3 wt%)	Nylon 450 nm filters	Menthol enhancer permeation studies, comparison with porcine ear skin and ATR-FTIR	FCA, PF, ALB, THC, and THP	PVPA model was adapted towards employment in a permeation-enhancing effect evaluation. The enhancement ratios obtained were in accordance with data from porcine ear skin. ATR-FTIR analysis detected a similar mechanism of menthol in both models (PVPA and porcine ear skin).	[193]
EPC:Chol (77:23 wt%)	Nylon 450 nm filters	SEM, electrical resistance, ATR-FTIR, and permeation studies	FCA, PF, ALB, THC, and THP	Papp values obtained through the PVPA-based system were well correlated with the values obtained through porcine ear skin. Better relevance between porcine ear skin and the PVPA-based system was found compared to that between porcine ear skin and a commercial Strat-M® membrane.	[194]
Cer:EPC:Chol:SA:ChS (50:25:12.5:10:2.5 wt%)	Polycarbonate 400 nm filters	SEM, electrical resistance permeation, and stability studies	CAL, CAF, CSP, DCF, MTX, and NAP	Modified PVPA presented low permeability of hydrophilic markers when compared to the original PVPA (mimicking the intestinal barrier). Good data correlation with permeability studies in a pig ear model.	[195]
		Stability studies and permeability studies	CAL	Modified PVPA kept stable for 12 weeks when stored at −20 °C. Barrier integrity was not affected by the presence of a set of cosolvents, but ethanol altered its integrity.	[196]

_aCer—analog ceramides; AA—arachidic acid; ALB—albiflorin; APAP—acetaminophen; ATR-FTIR—attenuated total teflection Fourier-transform infrared; BA—behenic acid; BSC—bovine spinal cord; CA—cerotic acid; CAF—caffeine; CAL—calcein; CAP—chloramphenicol; Cer—ceramide; Cer[AdS]—α-hydroxy fatty acid/dihydrosphingosine base ceramide; Cer[AP]—α-hydroxy fatty acid/phytosphingosine base ceramide; Cer[EOdS]—ω-hydroxy fatty acid/dihydro-sphingosine base ceramide; Cer[EOS]—ester-linked ω-hydroxy fatty acid/sphingosine base ceramide; Cer[NdS]—non-hydroxy fatty acid/dihydrosphingosine base ceramide; Cer[NP]—non-hydroxy fatty acid/phytosphingosine base ceramide; Cer[NS]—non-hydroxy fatty acid/sphingosine base ceramide; Chol—cholesterol; ChS—cholesteryl sulfate; CIP—ciprofloxacin; CSP—cyclosporine; DCF—diclofenac; DDA—dodecyl acetate; DLS—dynamic light scattering; E-80—egg phospholipid lipoid E-80; EM—electron microscopy; EPC—egg phosphatidylcholine; ESEM—environmental scanning electron microscopy; FCA—ferulic acid; FFA—free fatty acid; Flu—flufenamic acid; FTIR—Fourier-transform infrared; FUR—furosemide; HC—hydrocortisone; _hCer—human isolated ceramide; IBP—ibuprofen; IND—indomethacin; IR—infrared; LA—lignoceric acid; MTX—methotrexate; NAP—naproxen; NFD—nifedipine; PA—palmitic acid; PABA—p-aminobenzoic acid; PAMPA—parallel artificial membrane permeability assay; PEF—pefloxacin; PF—paeoniflorin; PVPA—phospholipid vesicle-based permeation assay; _pigCer—pig isolated ceramide; SA—stearic acid; SAL—salicylic acid; SAXS—small-angle X-ray scattering; _synCer—synthetic ceramide; SEM—scanning electron microscopy; SPC—soybean phosphatidylcholine; SWAXS—small- and wide-angle X-ray scattering; TA—tricosylic acid; TEWL—transepidermal water loss; TH—theophylline; THC—tetrahydrocolumbamine; THP—tetrahydropalmatine; WAXS—wide-angle X-ray scattering; VER—verapamil; XRPD—X-ray powder diffraction.