Materials
Tryp (≥ 98% purity) and LGS were purchased from Yuanye Bio-Technology (Shanghai, China). 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol] (DMPG) were obtained from Avanti Polar Lipids, Inc. Propanediol, triethanolamine, ethyl acetate and carbopol were supplied by Aladdin Biochemical Technology (Shanghai, China). Methanol (HPLC and MS grade), acetonitrile (MS grade) and isopropanol (HPLC grade) were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA). Methyl tert-butyl ether (MTBE), formic acid (HPLC grade) and ammonium formate (HPLC grade) were obtained from Sigma-Aldrich (St. Louis, Mo, USA).
Synthesis of Tryp-loaded ethosomes and Tryp-loaded ethosome gel
Tryp-ES were synthesized using a polydimethylsiloxane (PDMS) microfluidic chip, which comprises three inlets and one outlet (Wenhao Co. Ltd, Suzhou, Jiangsu, China). Briefly, Tryp-ES were prepared by injecting into the middle channel of the microfluidic chip a mixture of DMPC (5–15 mM) with Tryp dissolved in propanediol (0–100 µg/mL) at a rate of 200–400 µL/min. Solutions of LGS in double-distilled water (ddH2O, 0.01–0.03% w/v) were injected into the two external channels at a rate between 300 and 400 µL/min for the optional strategy. The resulting products were collected from the outlet channel.
Gel carrier of empty ethosomes or Tryp-loaded ethosomes (Tryp-ES gels) were prepared using 1.5% (w/v) carbopol, 5% (w/v) propanediol, 1.5% (w/v) triethanolamine, and distilled water with or without Tryp-ES consisting of 0.00075% (w/v) Tryp. Initially, a homogeneous solution consisting of distilled water, ES or Tryp-ES solutions, and propanediol was prepared in specific proportions. When preparing various doses of ethosome gels, the total volume of the solution was adjusted to 3, 6, and 9 mL, respectively. Carbopol powders were then evenly dispersed into the solution and allowed to swell gradually and uniformly overnight at 4 °C. The following day, triethanolamine was added to neutralize the carbopol and achieve a pH of approximately 5.3 to facilitate stable gel formation. Simultaneously, the mixture was vigorously stirred to ensure even distribution of the triethanolamine and initiate the gelation process, yielding the empty ethosomes gels and Tryp-ES gels.
Determination of particle size, polydispersity index (PdI) and zeta-potential
The particle size (weighted by intensity), PdI and zeta-potential of the prepared ethosome formulations were measured using Malvern Zetasizer Ultra (Malvern Panalytical Ltd., Malvern, UK) at 25 °C. All the batches were diluted with Milli-Q ultrapure water (Millipore, Molsheim, France) at 1:10 dilution and analyzed. All measurements were performed in three parallel replications.
Transmission electron microscopy (TEM)
The morphology of the ethosomes or Tryp-ES was characterized by transmission electron microscopy (TEM). For TEM, 10 µL samples were added to a carbon-coated copper grids (Beijing Zhongxing Bairui Technology Co., Ltd., Beijing, China), then negatively stained with 10 µL 2% sodium phosphotungstate (Yuanye Bio-Technology, Shanghai, China). Once stained and air-dried, the images were taken with a Hitachi H7650 TEM microscope (Tokyo, Japan) at 80 kV.
Atomic force microscopy (AFM)
The surface topography of prepared ethosomes was analyzed by AFM Cypher ES (Asylum Research, Oxford Instruments, Santa Barbara, CA, USA). Briefly, samples were dropped onto 10 mm mica discs (TedPella, USA) and vacuumed dry at room temperature (RT) for 2 h. Scanning was performed in AC Air Topography mode using AC200TS-R3 probe (Olympus, Japan) with a constant of 9 N/m at a temperature of 25 ± 1 °C.
The influence of prepared ethosomes on the morphology of DMPC and DMPG (4:1) bilayers was examined by AFM Cypher ES. A solution of DMPC (2 mM) and DMPG (0.5 mM) in anhydrous ethanol was quickly mixed and sonicated for 30 min at RT. The mixture was deposited onto freshly cleaved mica and incubated at 40 °C for an hour. The surface was rinsed three times, and the bilayers were imaged in the buffer by the addition of 800 µL ethosomes over the supported bilayers. Scanning was performed in AC Water Topography mode using an AC160TSA-R3 probe (Olympus, Japan) with a spring constant of 26 N/m and a resonance frequency of 300 kHz. The membranes samples were scanned at a rate of 1.7 Hz, with a drive frequency of 125 ± 5 kHz, and a resolution of 512 × 512 pixels. The images were analyzed using Asylum Research Real Time software (v.18.04.23).
High-performance liquid chromatography (HPLC)
The concentration of Tryp in the samples was quantified by an Agilent 1100 HPLC system equipped with a UV detector (Agilent Technologies, Palo Alto, CA, USA). In chromatography, the C18 column (Agilent, Stable Bond 300, 250 mm × 4.6 mm, 5 μm) was used with a methanol/ddH2O mobile phase (60:40, v/v) at a flow rate of 1 mL/min. The amount of Tryp was quantified at 254 nm, with a retention time of 5.5 min. The peak of Tryp was successfully distinguished from other peaks in the chromatogram, demonstrating no interference from the other peaks. The concentration of Tryp was determined by means of a calibration curve spanning the range from 0.01 to 10.0 µg/mL. The same concentration solutions of Tryp standards were subjected to analysis on three consecutive occasions using the established HPLC method. In all cases, the relative standard deviation (RSD) was less than 3.0%, which fulfills the technical requirements for an HPLC chromatogram.
Entrapment efficiency (EE)
The prepared samples were centrifuged at 1000 rpm, 4 °C for 2 min to separate the unencapsulated drug. The supernatant was collected and then treated with 0.1% Triton X-100 (Sigma) to disrupt the ethosomes. All samples were filtered through a 0.45 μm membrane (Millipore, Billerica, MA, USA) before HPLC analysis. The entrapment efficiency (EE) was calculated according to the following equation:
$$\text{Entrapment efficiency }\left(\%\right)\hspace{0.17em}=\hspace{0.17em}\text{Ts/Tp}\times100,$$
where Ts is the content of Tryp loaded in the formulation, and Tp is the initial content of Tryp added in the formulation.
Stability evaluation of ethosomes
Tryp-ES were stored at 4 ± 1 °C or 25 ± 1 °C (RT) for 28 days. Both the physical and the chemical stability of Tryp-ES were evaluated. The physical stability was assessed by visual observation, particle size, PdI, and zeta potential determination. The chemical stability was determined by measuring the content of Tryp encapsulated in ethosomes by HPLC on days 1, 7, 14, 21 and 28. The physical evaluation of gel appearance, pH, and drug content was conducted over a period of 28 days at 4 °C and 25 °C for all prepared gel formulations. The pH of the Tryp-ES gel was determined using a Sartorius PB-10 pH meter (Sartorius, Germany). Briefly, 1 g of Tryp-ES gel was dissolved in 100 mL of ultrapure water, and the solution was tested in triplicate, with the average value calculated. Prior to analysis of the drug content, the gels were diluted with ultrapure water in a 1:100 ratio (gel: ultrapure water = 1:100). The drug content was analyzed by HPLC, as described above.
In vitro drug release
The Tryp release profile from Tryp solution, Tryp-ES and Tryp-ES gel were assessed using the dialysis bag (3500 MWCO, Biorigin, Beijing, China) diffusion method in a 40 mL solution with 10% propanediol at 32 ± 1 °C. For a period of 48 h, 1 mL of solution was collected from each sample at 10, 20, 30 min and 1, 2, 4, 8, 12, 24, 48 h, while 1 mL fresh solution was added to each sample to maintain a constant total volume of 40 mL. The amount of released Tryp was analyzed by HPLC.
Skin permeation and retention studies
The in vitro skin permeation and retention of Tryp from Tryp-ES were investigated using Franz type glass diffusion cells as previously described in detail [31]. Specifically, the dorsal skin of psoriatic mice treated with 62.5 mg IMQ cream daily on the exposed dorsal skin for 7 days was excised, the subcutaneous fat was carefully removed, and the skin was mounted with the SC facing up on a Franz type glass diffusion cell (Huke, Jiangsu, China) with an effective area of 3.14 cm². Briefly, excised psoriatic mouse skin was mounted between the donor and receptor compartments with the dorsal surface of the skin facing up into the donor chamber. Tryp solution, Tryp gel, Tryp-ES or Tryp-ES gel were applied onto the skin in the donor chamber without sealing. The receptor chamber was filled with 10% propanediol and stirred with a magnetic bar at 200 rpm to equilibrate at 32 ± 1 °C. At time intervals of 0, 10, 20, 30, 40, 50 and 60 min, or 2, 4, 8, 12 and 24 h, a 1 mL aliquot of receptor was collected, and the same volume of fresh medium was added back. The amount of Tryp in the samples was analyzed by HPLC, and the cumulative amount was plotted against time. The permeation of drug was calculated according to the following formula.
Permeation of drug (%) = (amount of permeated drug/initial amount of drug) × 100.
For the drug retention assay, skin samples were collected from the donor chamber at 1 h, or 2, 4, 8 and 24 h. The epidermis was separated from the dermis under the condition of 0.125–0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) at 25 or 37 °C for 0.5–2 h (Table S1) and tissue samples were ground using a cryogenic grinder (JXFSTPRP-II, Jingxin Industrial Development Co., Ltd., Shanghai, China). After extraction of Tryp from skin tissues with ethyl acetate three times, the samples were concentrated under vacuum. The precipitate was dissolved in 200 µL DMSO (Aladdin) for further HPLC analysis.
Skin biodistribution studies
Rhodamine B (0.01%, w/v, Sigma-Aldrich) loaded ethosomes (RhB-ES) were prepared using the microfluidic device described in the synthesis of Try-ES. The method used for preparing Rhodamine B ethosomal gel (RhB-ES gel) was identical to that used for Tryp-ES gel. RhB-ES or RhB-ES gel with the same dose of rhodamine B was applied topically to the dorsal skins of IMQ mice. At 0, 10, 20, 30 and 60 min, the back skin of the mice treated with RhB-ES and RhB-ES gel was cut off and embedded in the optimal cutting temperature compound (Sakura Finetek, Torrance, California, USA). After being snap-frozen in liquid nitrogen, the tissue was sectioned at 8 μm. The penetration and biodistribution of RhB-ES and RhB-ES gel of different skin layers were evaluated by confocal laser scanning microscopy (Olympus FV3000, Tokyo, Japan).
Cell culture
Human keratinocytes (HaCat) were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S, Gibco). Human umbilical vein endothelial cells (HUVEC) were cultured in complete endothelial cell medium (ECM, ScienCell, San Diego, CA, USA) supplemented with 10% FBS (ScienCell), 1% endothelial cell growth supplement (ECGS, ScienCell) and 1% P/S (ScienCell). All cells were cultured in a humidified 5% CO2/37 °C incubator. Cells were regularly passaged every 5–7 days with 0.25% trypsin-EDTA (Gibco).
Cell proliferation
Cell proliferation was assayed using CellTiter96® Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). Briefly, HaCat cells were seeded at a density of 1.5 × 104 cells/cm2 and cultured with growth medium supplemented with EGF or KGF (0–160 ng/mL) (Abcam, Cambridge, MA, USA) and various concentrations of Tryp solution or Tryp-ES as indicated for 24 h. The cells were then washed by replacing the culture medium with MTS reagent for 3 h. 100 µL of the solution was transferred into a 96-well plate, and the absorbance was measured at 490 nm. The same volumes of culture medium and MTS reagent without cells were also incubated as the background.
In vitro cellular uptake
Cellular uptakes of Tryp-ES or Tryp solution was assessed using the intrinsic fluorescence properties of Tryp with a fluorescence microscope. HaCat cells were seeded in 12-well plates at a density of 6.5 × 103 cells/cm2. When the cells confluence reached 80%, they were washed and incubated with Tryp-ES or Tryp solution at an equivalent Tryp dose of 300 ng/mL for 1, 3, 6, 12 and 24 h. The cells were then washed three times and fixed with 4% paraformaldehyde (Solarbio, Beijing, China). The samples were permeabilized with 0.2% Triton X-100 and blocked with 1% bovine serum albumin (BSA, Sigma) in PBS for 1 h. F-actin and nuclei were stained with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific) and DAPI (Abcam), respectively. After washing with PBS, the red fluorescence of Tryp in cells was visualized using the confocal laser scanning microscopy (Olympus) with a 100 × oil immersion objective (numerical aperture 1.4) at 568 nm laser. The z-stack images were then processed as mean intensity projection using FV31S-SW software (v.2.3.2, Olympus).
TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using CoraLite® Plus 488 TUNEL Apoptosis Detection Kit (Proteintech, USA) according to the manufacturer’s recommended protocol. HaCat cells were seeded at a density of 2.5 × 104 cells/cm2 on 24-well plate and incubated overnight to reach 60% confluence. Then, cells were treated with EGF (20 ng/mL) and various concentrations of Tryp-ES or Tryp solution as indicated for 24 h. Following fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100, the TUNEL reaction solution was added and incubated at 37 °C in the dark for 1 h. Before detection, cells were washed three times and counterstained with DAPI to label all nuclei. The fluorescence in the cells was observed using a confocal laser scanning microscope (Olympus).
Tube formation assay
To analyze the angiogenic activity of Tryp-ES, the extracellular matrix (ECM) gel-based capillary tube formation assay was used. This was performed following the manufacturer’s instructions. Briefly, HUVECs at a density of 7 × 103 cells/well were seeded on top of matrigel (R&D Systems, MN, USA) coated angiogenesis µ-slides (Ibidi GmbH, Gräfelfing, Germany) at 37 °C. The cells were applied with the treatment of 50 ng/mL recombinant human FGF-basic (bFGF, R&D) or various concentrations of Tryp-ES as indicated in the presence of bFGF for 6 h at 37 °C in a humidified atmosphere with 5% CO2. Cells were stained with Alexa Fluor 488 Phalloidin and DAPI, and the level of capillary tube structure formation was photographed using Zeiss Axio Observer microscope (Göttingen, Germany) and quantified via the AngioTool (v 0.6a, 64 bits) analysis software.
In vivo animal study
BALB/c mice (male, 6–8 weeks of age, 20 ± 2 g) were purchased from Beijing Vital River Laboratory Animal Technology CO., Ltd. (Beijing, China). All animals were group-housed together with free access to food and water in a controlled experimental environment with a consistent temperature/humidity and a standard light/dark cycle. All animal experiments were performed in compliance with the National Guidelines for Care of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences.
IMQ, a TLR7/8 ligand and potent immune activator, produces a psoriasis-like cutaneous phenotype in mice, frequently studied as the model of human psoriasis. The mice were divided into 15 groups mice (n = 3): one group is normal (non-treated group), one group was treated with IMQ (model group) to create the prosiasis-like condition, and the remaining groups were also treated with IMQ but given various treatments, including clobetasol propionate (CP, positive control group), single, double and triple dosing of ethosome vehicle (EV1, EV2, EV3), and single, double and triple dosing of Tryp-ES (Tryp-ES1, Tryp-ES2, Tryp-ES3), as well as those formulations in gel (EV1 gel, EV2 gel, EV3 gel, Tryp-ES1 gel, Tryp-ES2 gel and Tryp-ES3 gel). The ethosome vehicle contained 1 µM DMPC, while the Tryp-ES contained 1.5 µg Tryp loaded into the ethosome vehicle as a single dose. The model group was induced by local administration of 62.5 mg IMQ cream to the dorsal skin once every day for 7 days. Based on the response and tolerability of mice to the tested formulations in pilot studies, ethosome solutions or gels were topically applied to the lesion area of the skin one to three times a day for 20 min each time. The mice were weighed daily, and photographs of the dorsal skin were captured. On the 8th day, all groups were humanely euthanized, and the dorsal skin, liver, kidney, and spleen samples were collected. Blood was taken and stored at 4 °C overnight and then centrifuged at 3000 rpm, 4 °C for 15 min to obtain the serum. Levels of Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), globulin (GLB), creatine kinase (CK), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and creatinine (CR) were determined using automatic biochemical analyzer (TBA-40FR, TOSHIBA, Tokyo, Japan) to assess potential toxicity in the liver, heart, and kidneys.
Evaluation of the severity of psoriatic skin lesion
The severity of inflammation on the mice dorsal skins was assessed using the clinical Psoriasis Area and Severity Index (PASI), an objective scoring system was used to evaluate the severity of inflammation [32]. Erythema and scaling scores were obtained daily by blindly scoring with a scale from 0 to 4 (0-none; 1-slight; 2-moderate; 3-marked; 4-very marked). The scores for the scaling and erythema were combined to calculate a total score ranging from 0 to 8, which was assessed once daily for 7 days post-administration.
Hematoxylin and eosin (H&E) staining
Fresh tissues (skin, liver, spleen and kidney) collected from mice were fixed in 4% paraformaldehyde for 48 h. Samples were sequentially subjected to gradient ethanol dehydration and transparent treatment in Histo-Clear (National Diagnostics, GA, USA). Then, the samples were embedded in paraffin, and sectioned into 4-µm slices using a microtome (Leica, Nussloch, Germany). After dewaxing and rehydration, the tissue sections were stained with hematoxylin-eosin staining solution (Solarbio) to identify tissue cytoplasm structures (pink) and distinct nuclei (blue-violet). Images were captured using the Aperio Versa 8 tissue imaging system (Leica, Germany) with an image analysis system (Aperio V.12.4).
Immunohistochemistry (IHC)
For IHC staining, the hydrated sections were subjected to antigen retrieval in citrate buffer (Beyotime, Shanghai, China) at 95 °C for 15 min and cooled down until RT. After inactivating endogenous peroxidase, nonspecific binding sites were blocked with 5% goat serum. Samples were incubated with primary anti-Ki67 (ab16667, 1:400), anti-CD31 (ab124432, 1:1000), anti-CD3 (ab16669, 1:500) from Abcam overnight at 4 °C, followed by incubation with goat anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) for 30 min at RT. The positive staining was detected by using diaminobenzidine (DAB) (Zhongshan Golden Bridge Biotechnology) and the nuclei were counterstained with hematoxylin. Images were captured by Aperio Versa 8 scanner and analyzed by ImageJ software v 1.53a (NIH, Bethesda, MD, USA).
Lipidomic analysis
Lipids were extracted from mouse skin tissue using the MTBE method. Briefly, 300 mg of skin tissue was weighed and homogenized with 200 µL water and 240 µL methanol. Then, 800 µL MTBE was added and the mixture was sonicated at 4 °C for 20 min, followed by 30 min exposure at RT. The solution was centrifuged at 14,000 × g for 15 min at 10 °C, and the upper organic solvent layer containing the lipids was collected and dried under nitrogen.
The LC-MS analysis was performed with UHPLC Nexera LC-30 A (Shimadzu, Japan) equipped Q Exactive Plus High-Resolution Mass Spectrometer (Thermo Scientific, USA). For UPLC analysis, a CSH C18 column (1.7 μm, 2.1 × 100 mm, Waters, USA) was used. The lipid extracts were re-dissolved in 200 µL 90% isopropanol/acetonitrile, then centrifuged at 14,000 × g for 15 min to obtain the supernatant. A linear gradient consisting of acetonitrile-water (60:40, v/v) with 0.1% formic acid and 0.1 mM ammonium formate (mobile phase A) and acetonitrile-isopropanol (10:90, v/v) with 0.1% formic acid and 0.1 mM ammonium formate (mobile phase B) was used with a gradient program. The injection volume was 3 µL, the flow rate was 300 µL/min, and the column temperature was maintained at 45 °C. Mass spectra were acquired by Q Exactive Plus in positive and negative mode, respectively. Detailed ion source (ESI) parameter settings included: Heater Temperature 300 °C, Sheath Gas Flow rate 45 arb, Aux Gas Flow Rate 15 arb, Sweep Gas Flow Rate 1 arb, spray voltage 3.0 kV, Capillary Temp 350 °C, S-Lens RF Level 50%, MS1 scan ranges: 200–1800. The mass spectrometry data were collected using LipidSearch v. 4.2 (Thermo Scientific, USA) software. After analysis, a data matrix containing lipid molecule identification and quantification was obtained.
The original data were further analyzed and filtered using Python (v. 3.12, Python Software Foundation). To obtain group clustering, supervised partial least squares discriminant analysis was performed on the data. Fold change (FC) was detected using univariate analysis and t-test analysis was performed to calculate P value. To identify patterns and significant differences in lipid metabolite expression profiles among the different treatment groups, principal component analysis (PCA) and volcano plots were generated using the scikit-learn library (v. 1.4.1) and matplotlib (v. 3.8.2). Differential metabolites between the two groups in volcano plots were based on VIP > 1, FC > 1.5 or < 0.67, and p < 0.05.
Statistical analysis
Data are expressed as mean ± the standard error of the mean (SEM) and were evaluated using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Statistical significance was defined as p < 0.05. All statistical analyses were processed and figures were generated using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA).
