Dendrimer-conjugated isotretinoin for controlled transdermal drug delivery | Journal of Nanobiotechnology

Materials and reagents

Isotretinoin was obtained from APExBIO Technology (Houston, TX, USA) and applied to cells under dimmed yellow light. Advanced MEM, phosphate-buffered saline (PBS, 10 ×), antibiotic–antimycotic solution, fetal bovine serum (FBS), and l-glutamine solution (100 mM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The SZ95 human sebaceous gland cells [25] were cultured in Dulbecco’s Modified Eagle Medium (Gibco, USA), containing 10% FBS (Gibco, USA), 100 U/mL penicillin and streptomycin (Gibco, USA) in an incubator at 37 ℃ under 95% air and 5% CO2. Imiquimod cream was purchased from Sichuan Mingxin Pharmaceutical (Chengdu, China). The hydrophilic oligoether dendrimer was synthesized as reported previously [26,27,28,29].

1H-NMR measurement

An FT-NMR spectrometer was used to obtain 1H NMR spectra at 500 MHz. We used parts per million (ppm) to express the chemical shifts compared to the tetramethylsilane or residual solvent peaks (CDCl3:1H, 7.26). The indices s (singlet), d (doublet), and t (triplet) represent the multiplicity. Hz indicated the coupling constants.

Transmission electron microscopy (TEM)

To examine the self-assembled constructions, one drop of each sample solution was placed on a 200 mesh carbon-coated copper grid with Formvar (Carbon Type B [15–25 nm]; Ted Pella, Inc.) and then evaporated under surrounding conditions. We then performed negative staining of samples by depositing a drop of uranyl acetate aqueous solution (0.2–0.4 wt %) on the grid surface of loaded samples. For positive staining, the sample-loaded grids were exposed to RuO4 vapor, generated by reacting a mixture of RuCl33 H2O (0.2 g) and 5% NaClO (10 mL) for 5 min. A JEOL JEM HR2100 instrument operating at 120 kV was used to observe the dried specimens.

Analysis of dendrimer toxicity

Dendrimer toxicity was evaluated using Cell Counting Kit-8 (CCK-8). Prepared SZ95 sebocytes were treated with gradient concentrations of the dendrimer (0.025, 0.05, 0.1, 0.2 or 0.4 mg/mL) for 24 h. Next, CCK-8 reagent was added to the plates and incubated with cells for 1 – 2 h at 37 ℃ and under a humidified 5% CO2 atmosphere. Finally, absorbance was measured at 450 nm using a plate reader.

Synthesis of 13cRA-D

A mixture of 300 mg isotretinoin (13cRA; 1 mmol), 290 mg dendrimer (0.3 mmol), 45 mg 4-dimethylaminopyridine (0.37 mmol), and 82 mg dicyclohexylcarbodiimide (0.4 mmol) was maintained under stirring in dry toluene (8 mL) at 0 ℃ for 1 h. The reaction was performed for 3 days. After completion of the reaction, the reaction mixture was quenched with water and extracted using ethyl acetate following thin-layer chromatography. The brine used to wash the organic layer was then dried over anhydrous MgSO4. After the filtrate was condensed under reduced pressure, the crude product was purified by silica gel flash column chromatography using methanol:ethyl acetate (1:50) as the eluent to yield a 26% (100 mg) yellow liquid.

In vitro release study

We analyzed 13cRA-D release in vitro using a dialysis membrane (molecular weight cutoff = 1000 kDa). 13cRA-D was sealed in a dialysis bag and immersed in methanol (pH 5.0 or 7.0) at 37 ℃. Methanol was collected at 0.5, 1, 2, 4, 8, 12, 24 and 48 h. Then, methanol was measured using an ultraviolet–visible spectrophotometer to assess drug release. After the removal of each sample, 1 mL of fresh solvent was added to maintain a constant volume.

Ethical approval

Animal experiments were performed according to 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 permeation test

For transdermal testing, we employed the skin of Yorkshire pigs (1-month-old). We carefully excised the subcutaneous tissue to obtain an intact epidermis and then stored the skin at − 80 ℃ until use. The skin was cut into slices (approximately 30–30 mm2) and soaked in PBS at 37 ℃ for 1 h before use. Experiments were performed using a Franz diffusion cell at 37 ℃ and 100 rpm, avoiding light exposure. Then, 30% isopropanol and 70% PBS were added to receptor cells. The donor cells contained PBS, 0.25 mg/mL 13cRA, an equal weight of 13cRA-D comprising 0.25 mg/mL 13cRA or an equal weight of dendrimer. The prepared skin was placed between the receptor and donor cells. Samples were collected from the side arms at 2, 4, 6, 8, and 10 h to perform high-performance liquid chromatography (HPLC) measurements. All experiments were performed in triplicate.

Skin retention test

After observing skin penetration, skin samples were removed from the Franz diffusion cell, cut into small pieces of (~ 1 mm × 1 mm) and homogenized in a 20 mL mixture of chloroform:methanol (1:2) to extract the retained drug, which was measured using HPLC.

Drug distribution and retention

Following the skin penetration study, skin samples were cut into small pieces (~1 mm × 1 mm) and fixed in 4% paraformaldehyde overnight. Next, the samples were embedded in paraffin and sliced into approximately 4 μm tissue sections. Finally, mounted sections were observed under a fluorescence microscope, and images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Skin irritation test

The notum hair of 12 BALB/c mice (six males and six females, 20–30 g) was carefully removed, ensuring the epidermis remained intact. Mice were randomly divided into four groups treated with PBS, dendrimer, 13cRA or 13cRA-D, applied to the exposed skin; the applied substance was carefully removed with cotton after 6 h. Finally, the skin was scored (0–4) according to a published standard for evaluation [30]: 0, no erythema; 1 slight erythema; 2 moderate erythema; 3 moderate-to-severe erythema; 4, severe erythema.

Treatment effect in acne model

To construct the Kligman acne model [31], we applied coal tar to the ear tubes of 12 of 15 New Zealand white rabbits (males, 2–2.5 kg) once daily for 14 days. On day 15, the rabbits were randomly divided into four groups, which were administered PBS, dendrimers, 13cRA or 13cRA-D respectively, once daily until day 28. Next, we evaluated the gross morphological and histological changes. Acne severity was evaluated using the above-scored standards. We performed hematoxylin and eosin (H&E) staining to assess the development of hair follicle horn plugs.

Psoriasis model

We randomly divided 20 female BALB/c mice (20–30 g) into four groups and removed notum hair using a depilatory cream. Imiquimod cream (Sichuan Mingxin Pharmaceutical, Chengdu, China) was evenly applied daily to 15 of the 20 mice for seven days. We applied gentle pressure for 30 min until the cream was fully absorbed. Six hours after each application, PBS, 13cRA or 13cRA-D was applied for 30 s. Subsequently, we assessed the skin for gross morphological and histopathological changes. Skin inflammation severity was assessed using the Psoriasis Area Severity Index (PASI) [32]. Subsequently, we adjusted the mouse model based on evaluation using the clinical PASI score. The affected skin area was not considered in the overall score evaluation. Erythema and scaling were evaluated using a scale of 0 to 4 as follows: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. Pathological changes in the psoriasis-like model were observed using H&E staining and Baker scoring, ranging from 0 to 10, under a light microscope [33].

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using analysis of variance (ANOVA) or two-sample Student’s t-tests, followed by the least significant difference post hoc test. A p-value < 0.05 indicated a statistically significant difference between groups.