Cell lines and antibodies
Human umbilical cord mesenchymal stem cell lines (hMSCs) were provided by the Scientific Research Centre of China-Japan Union Hospital, Jilin University (Changchun, China). Isotretinoin was purchased from APExBIO Technology LLC (Houston, TX, USA). Immunohistochemical antibodies against IL-8 and TNF-α were purchased from Bioscience Tec LLC (Tianjin, China). Antibodies against clathrin heavy chain were purchased from Abcam Tec (Shanghai, China). Antibodies against keratin-10, claudin-1, ZO-1, and caveolin-1 were from Affinity Biosciences (Cincinnati, OH, USA). Antibodies against GAPDH and Horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit were provided by Santa Cruz Biotechnology (Danvers, MA, USA). Enhanced chemiluminescence reagent was obtained from Pierce Biotechnology (Rockford, IL, USA).
Preparation of STCM-ATRA-NPs
HMSC membrane was prepared following published protocols . Stem cells were lysed using hypotonic solution , followed by freeze-thaw disruption and differential centrifugation to get the purified stem cell membrane . To find the best way of combine the stem cell membrane with Isotretinoin (ATRA) , we tried different methods, and adjusted the ratio between STCM and ATRA . ATRA was first dissolved in alcohol solution into 20mg/ml , then added PBS solution into 4 mg/mL, 3 mg/mL, 2 mg/mL, 1 mg/mL, or 0.5 mg/mL. About 4 × 106 stem cell derived stem cell membranes in PBS(1ml) were used , subsequently, added 1ml isotretinoin-alchohol/PBS solution , the mixture was placed on a constant temperature oscillator at 37 °C for 24 h and 48 h, avoiding light. Alternatively, using the Ultrasonic method, after thawing , 4 × 106 stem cell derived stem cell membranes in PBS(1ml) was placed in an ultrasound cleaner for 5 min , the isotretinoin-alchohol/PBS solution was mixed with the stem cell membrane solution and placed in the ultrasound cleaner for 10 min. The stem cell-coated isotretinoin was separated from uncoated isotretinoin by centrifugation at 15000 ×g for 30 min at 4 °C, keeping the supernatant for UV mesurenment.
Characterization of STCM-ATRA-NPs
Transmission electron microscopy
The morphology of the nanoparticles was observed by transmission electron microscopy (TEM; JEM-1230, JEOL, Tokyo, Japan). The samples were dispersed directly into bi-distilled water. A drop of the STCM-ATRA-NPs suspension was transferred to a 300-mesh carbon-coated copper grid. After staining with 2% (w/v) phosphotungstic acid solution and drying at room temperature, the sample was observed by TEM at 70 kV.
Determination of encapsulation efficiency of stem cell membrane-loaded isotretinoin
To calculate encapsulation efficiency, after extruded from a 200 nm filter membrane , the amount of uncoated isotretinoin was measured by UV absorbance spectrophotometrically at λ = 355 nm. The encapsulation efficiency was relative to the original drug added, applying the following equation: Encapsulation efficiency = total drug amount - unloaded drug amount/total drug.
STCM-ATRA-NPs size analysis
The freshly prepared STCM-ATRA-NPs dispersion was diluted with double diluted water, and the nanoparticles were extruded from a 400 nm filter membrane. Following, a Dynamic Light Scattering particle size distribution analyzer (DLS , Brookhaven BI9000AT , New York , USA) was used to characterize the vesicle size and size distribution. The vesicle size range was set between 0.1 and 20 mm.
Zeta potential of STCM-ATRA-NPs
Zeta potential, an indicator of stability of the STCM-ATRA-NPs dispersion, was determined using Malvern instruments (Osaka, Japan).
Ultra-violet spectrophotometry of STCM-ATRA-NPs
An equal ATRA concentration of STCM-ATRA-NPs and STCM was sent for Ultra-violet spectrophotometry measurement at λ = 355 nm.
In vitro release study
Protected from light, isotretinoin release from the stem cell membrane was performed using the Franz diffusion cell (RYJ-6B, HuangHai, Shanghai, China). These cells consisted of donor and receptor chambers separated by dialysis tubing with a molecular weight cut-off of 12000–14000 (Spectrum Medical Inc., Los Angeles, CA, USA). The receptor cell was filled with 50% alcohol/50% PBS, and 1.0 mL/0.1 mg/mL STCM-ATRA-NPs was added to the donor cell. Samples were removed from the side arm at 0.5, 24, 48, 72, 96, 120, 144, and 168 h, and an equal volume of blank solution was added to the receptor cell. Isotretinoin concentration in samples was measured by ultra-violet spectrophotometry at λ= 355 nm. Measurements were carried out in triplicate.
Skin Permeation Test
Isotretinoin release was measured as described above. Briefly, a 1-month-old Yorkshire pig was sacrificed for its skin, and the subcutaneous fat was removed carefully to keep the stratum corneum intact. All animal experiments were performed in accordance with the guidelines for the Care and Use of Experimental Animals of Jilin University and were approved by the Animal Experiment Ethics Committee of Jilin University. Skin was kept at -80 °C before use. Further, skin was soaked in PBS for 1 h at 37 °C, and the skin was cut into small pieces (about 30 ´ 30 mm square samples) and placed between the receptor and donor cell. Additionally, 0.25 mg/mL ATRA (1 mL) and an equal weight of STCM-ATRA-NPs containing 0.25 mg/mL ATRA (1 mL) were added to the donor cell. An equal volume of stem cell membrane was added as the control group. The receptor cell was filled with 50% Isopropanol and 50% PBS without isotretinoin. Samples were taken from the receptor cell at 0.5, 1, 2, 4, 6, and 8 h at a volume of 0.5 mL, filtered, and quantified by HPLC (Acchrom S6000, Huapu Tec Inc., Beijing, China). An equal volume of receiving chamber solution without isotretinoin was added to the receptor cells. All studies were run at 37 °C, 100 rpm, avoiding light. Each experiment was repeated in triplicate.
Skin Retention test
The amount of drug retained in the skin samples was measured in permeation studies. After a skin penetration test, the skin was removed from the receptor cell, washed with PBS three times, and cleaned to remove any adhering formulation. Following, the skin was cut into small pieces, homogenized with 20 mL of chloroform: methanol at a volume ratio of 1:2, and vortexed for 10 min. After filtration using a 400 nm filter membrane, the drug content was measured by HPLC (Acchrom S6000).
Observation of drug distribution and retention by fluorescence microscopy
Briefly, 1-month Yorkshire pig skin was cut into small pieces and set between the receiving and donor chamber of the Franz diffusion cell. The receptor cell was filled with 50% Isopropanol and 50% PBS without Isotretinoin. Following, PBS, 0.25 mg/mL ATRA, STCM, and an equal volume of STCM-ATRA-NPs containing 0.25 mg/mL ATRA (1 mL) were added to the donor chamber. Skin was treated for 8 h at 37 °C, 100 rpm, avoiding light. Skin tissue was fixed with 4% paraformaldehyde overnight and embedded in paraffin. Fluorescence microscopy was conducted to observe the fluorescence distribution, and ImageJ software was used to quantify fluorescence.
Skin irritation test
Briefly, 12 New Zealand white rabbits were randomly assigned to four experimental groups: normal control, ATRA, STCM, and STCM-ATRA-NPs. PBS, 0.25 mg/mL ATRA cream, an equal volume of STCM, and an equal weight of ATRA-STCM-NPs containing 0.25 mg/mL ATRA were applied once to each group at a dose of 0.5 g. After 6 h, the test area was cleaned to observe visible changes. The scores were recorded (0–4) according to evaluation standards , with 0 indicating no erythema, 1 = slight erythema, 2 = moderate erythema, 3 = moderate-to-severe erythema, and 4 = severe erythema.
Treatment effect in follicular hyperkeratosis model
Male New Zealand white rabbits (2–2.5 kg; Changchun Biological Products Co. LTD) were housed 1 per cage under a light/dark cycle of 12 h with free access to water and food continuously for 5 weeks. To establish the follicular hyperkeratosis model, 15 rabbits were randomly assigned into five groups: normal control (blank), hyperkeratosis+PBS (vehicle), ATRA, STCM, and STCM-ATRA-NPs. Further, 12 rabbits were given coal tar once a day on the ear tube (about 2 × 2 m²) for 14 days to establish the Kligman acne model. Based on established acne criteria, 0 indicates no erythema; 1 = slight erythema, comedo; 2 = moderate erythema, comedo; 3 = moderate-to-severe erythema, comedo; and 4 = severe erythema, comedo. Histological changes were evaluated by H&E staining and IHC staining.
Treatment effect in hyper sebum production model
Male golden hamsters (6–8 weeks, 80–120 g) were used to establish a hyper sebum production model. Hair was removed carefully to expose the two sides of the sebaceous glands, and the hamsters were randomly divided into four groups: normal control (blank), ATRA, STCM, and STCM-ATRA-NPs. Formulations were given once daily, and the animals were sacrificed on day 14. Histological changes were evaluated by H&E staining.
Scanning electron microscopy and Fourier infrared spectrum analysis
Briefly, 1-month-old Yorkshire pig skin was processed in the Franz diffusion cell, as described above. The receptor cell was filled with 50% Isopropanol and 50% PBS without Isotretinoin. Further, PBS, 0.25 mg/mL ATRA, STCM, and an equal volume of STCM-ATRA-NPs containing 0.25 mg/mL ATRA (1 mL) were added to the donor chamber. Skin was treated for 8 h at 37 °C, 100 rpm, avoiding light. For SEM analysis, the skin was fixed with glutaraldehyde for 24 h, freeze-dried for 48 h, and observed using a scanning electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) to assess changes in the Horney layer of the skin layer at 200× and 900× magnification. For Fourier infrared (FTIR) spectral analysis, the skin was removed from the freeze-drying machine after 48 h, ground with potassium bromide using an agate mortar, and analyzed at resolution: 2 cm-1, scanning times: 100, and scan range: 650–4000 cm-1 to assess changes in keratin.
Briefly, 1-month-old Yorkshire pig skin was processed in the Franz diffusion cell, as described above. After treatment for 8 h at 37 °C, 100 rpm, avoiding light, the skin was removed and ground in liquid nitrogen for protein extraction. Ice-cold lysis buffer was used to treat skin tissue samples , and the relative concentrations were determined by BCA protein kit. The supernatants of the cell lysates were then separated by 12 % SDS-PAGE gels and transferred onto PVDF membranes, which were subsequently blocked with 5% no-fat milk. Finally, the membranes were reacted with primary antibodies for 12 h, followed by keratin-10 , claudin-1 , ZO-1 , caveoline-1 and clathrin heavy chain secondary antibodies.
All data are given as mean ± standard error (SE). Statistical analyses were performed using two-sample Student’s t tests and ANOVAs, followed by an LSD post-hoc test. P < 0.05 was considered statistically significant.