Preparation of pro-retinal nanoparticles (PRNs)
PRNs were prepared by grafting retinal (Sigma-Aldrich) onto chitosan (CS, Mw of 40,000–50,000 Da, Taming Enterprise, Samut-Sakhon, Thailand) polymer. The grafting was carried out using our previously reported protocol . In brief, CS was dissolved in 0.1% acetic acid, and the pH of the obtained solution was adjusted to 5.9 using NaOH. The final solution contained 45 mg CS in 19.0 mL solution. The grafting reaction was initiated by slowly adding retinal (15 mg in 1.0 mL of ethanol) dropwise into the two-neck round-bottom flask containing aqueous CS suspension at 5°C, under ultrasonic (40 kHz), light-proof and N2 atmospheric condition. After stirring for 2 h, the pH of the solution was adjusted to 6.8, and PRN suspension was obtained. The particles size of PRN was examined by scanning electron microscopic (SEM, JSM-6400; JEOL, Tokyo, Japan).
Composite materials for microneedles
Binary mixtures of various polymers and sugar were experimented for microneedle fabrication. Hyaluronic acid (HA, injection grade, Shandong Focuschem Biotech Co., Ltd., Shandong Sheng, China) was mixed with each of the following materials: polyvinyl pyrrolidone (PVP K30, average Mw 40,000, Sigma-Aldrich, Germany), polyvinyl alcohol (PVA, average Mw 146,000-186,000, Sigma-Aldrich), carboxymethyl cellulose (CMC, Dow Chemical Thailand Ltd, Bangkok, Thailand), sericin (Seritech Co., Ltd, Bangkok, Thailand) and maltose (Sigma-Aldrich), at two ratios. Solution of selected materials was prepared in water and the obtained solution was used to prepare microneedle arrays via the molding method as previously described [31, 35]. The total of 11 formulations of needle matrix material were experimented and they include (expressed here as weight percentages) 100% HA, 50% PVP + 50% HA, 25% PVP + 75% HA, 50% maltose + 50% HA, 25% maltose + 75% HA, 50% PVA + 50% HA, 25% PVA + 75% HA, 50% CMC + 50% HA, 25% CMC + 75% HA, 50% sericin + 50% HA and 25% sericin + 75% HA. Microneedles used in mechanical testing and stability experiments were 8×8 mm patch containing an array of 10×10 needles of the tetragonal pyramidal shape with 300×300 µm base, 650 µm needle height, the tip-to-tip distance of 500 µm. The needles were sitting on a 500 µm thick base made of the same material as the needles.
For DMN patches that contain PRN or retinal, the preparation was the same except that PRN or retinal was added to the polymer solution. PRN-loaded microneedles were prepared by dissolving HA (40 mg) and maltose (40 mg) in the PRN suspension (3 mg CS, 1 mg retinal, 1 mL of 7% ethanol in water). The obtained mixture was loaded into the mold and let dry as described above. Retinal-loaded DMNs were prepared by dissolving HA (40 mg) and maltose (40 mg) into retinal solution (1 mg retinal, 1 mL 7% ethanol in water) and the obtained mixture was loaded into the mold and let dry as previously described.
All the obtained microneedles were examined under stereomicroscope (Olympus DP22, Japan). Mechanical testing was carried out on DMN patches using the universal testing machine (UTM, Shimadzu EZ-S, Shimadzu Corporation, Tokyo, Japan). Distribution of retinal and PRNs in the DMN patches were acquired by taking confocal fluorescence images (Confocal fluorescence microscope, Eclipse, Ti series microscope, Nikon, Japan) of each DMN patch at all layers and then stacking the obtained images into three dimensional images.
Detachable dissolvable microneedles for ex vivo and in vivo experiments
PRN-loaded detachable dissolvable microneedle (DDMN) patch was prepared by dissolving HA (40 mg) and maltose (40 mg) in the PRN suspension (3 mg CS, 1 mg retinal, 1 mL of 7% ethanol in water). The obtained mixture of 0.05 ml was loaded into the silicone mold, and the filled mold was put in the light-proof and moisture-controlled (~ 5%) chamber until dry. Then 0.05 ml of vicious polymer solution (without PRN) was dropped onto the dry microneedle array (in the PDMS mold) and the water penetrable lint-free sheet (600 µm thick woven polyester sheet) was attached, then the assembled piece was put in the light-proof and moisture-controlled (~ 2%) chamber until the array was completely dry. Retinal-loaded DDMN patch was prepared similarly except that the freshly made retinal solution was used in placed of PRN suspension. Retinal solution was prepared by dissolving 1 mg of retinal into 1 mL of 7% ethanol in water. The unloaded DDMN patch was prepared similarly except that water was used in placed of the PRN suspension. DDMN patches used in ex vivo experiment. The obtained DDMN patches were 8×8 mm patch containing an array of 10×10 needles of the tetragonal pyramidal shape with 300×300 µm needle base, 650 µm needle height, the tip-to-tip distance of 500 µm and were used for ex vivo drug diffusion experiments. DDMN patches used for in vivo skin irritation experiment were prepared similarly with adjusted amount of PRN (8 µg of retinal per patch), and the patch was 8×8 mm patch containing an array of 10×10 needles of the tetragonal pyramidal shape with 100×100 µm base, 150 µm needle height, the tip-to-tip distance of 500 µm.
Chemical stability of retinylidene moieties in PRN-loaded DMNs (amount of retinal = 50 µg per patch) and in PRN suspension (concentration of retinal = 1 mg/mL of the suspension), and stability of retinal in retinal-loaded DMNs (amount of retinal = 50 µg per patch) and in retinal solution (concentration of retinal = 1 mg/mL, solvent = 7% (v/v) ethanol in water), were examined.
Each DMN sample was kept in individual light-proof package at 4, 25, 40, and 50 °C. At each time point (0, 7, 14, 30, 60, and 90 days), the DMN sample was taken out and subjected to extraction and quantification. For retinal-loaded DMN or PRN-loaded DMN, the DMN patch was dissolved in 2 mL acidic water (0.0025 M HCl) under N2 atmosphere and light-proof condition. The solution was left for 10 min. Then the solution was partitioned with ethyl acetate (3 times with 2 mL ethyl acetate each time). The collected ethyl acetate extract was quantified for retinal by UV-vis spectrophotometry using maximum wavelength of 370 nm with the aid of a calibration curve.
For retinal solution or PRN suspension, the samples were kept under light-proof condition at the same temperatures and same durations. At each time point, the liquid sample was subjected to retinal extraction. The extraction was initiated by transferring 5 M HCl into 2 mL of the sample to reach the final HCl concentration of 0.0025 M. The mixture was left under N2 and light-proof condition for 10 min before subjecting to ethyl acetate partition (3 times with 2 mL ethyl acetate each time). The collected ethyl acetate extract was quantified for retinal as described above.
Ex vivo drug diffusion in skin tissue
Fresh porcine ear from local slaughterhouse (Lopburi, Thailand) was cleaned with citrate buffer (0.1 M, pH 7.4). The hairs were shaved, and the ear was rinsed with water and dried with tissue paper. The DDMN samples (650 µm of needles height) were pressed against the full-thickness porcine ear skin, then 1 drop of water was applied and the DDMNs were pressed for 1 min, then the backing sheet was removed. The ear was kept in a close petri dish lined with PBS buffer-soaked paper towel with the subcutaneous side touching the paper towel (to keep the tissue moist) for 0, 1 and 4 h. At each time point, the full thickness skin was surgically sectioned, and the tissue section was examined under stereomicroscope and confocal fluorescence microscope. Diffusion of retinoid in skin tissue pieces was examined under stereomicroscope and confocal fluorescence microscope (Eclipse, Ti series microscope, Nikon, Japan) using the excitation and emission wavelengths of 525 nm and 488 nm, respectively.
In vivo skin irritation
The study protocol was approved by the committee of the use of animal for the scientific purpose of Chulalongkorn University animal care (protocol no. 1873021). Eight male rats (Wistar rat, 10–12 weeks, 300–400 g, Nomura Siam International Co. Ltd., Thailand) were housed at Chulalongkorn University Laboratory Animal Center, in an isolated clean room held at 25 ± 2 °C, with a relative humidity of 65–75%. The rats were acclimatized for 3 weeks before starting the experiment. Rats were divided into two groups of 4 rats each: 24 h and 7 d groups.
For the treatment, rats were anesthetized and their hairs at the treated area were shaved. Each rat was treated with PRN-loaded DDMN patch (equivalent to 8 µg retinal per patch) on the right dorsal skin, and unloaded DDMN patch on the left dorsal skin. The administration was carried out by pressing the DDMN patch against the skin using pressure from fingertips for 1 min, then one drop of water was dropped over the backing of the DDMN patch, and the patch was hold for another 1 min. After that, the base sheet of the DDMN patch was taken out. The treated area was observed at 1, 6, 12, 24 h and every day until day 7 post the administration. Full thickness skin of the test area was sampled at 24 h and 7 d post the single administration. The biopsied skin tissue was fixed in 10% formalin buffer and processed as routine histopathological techniques. The skin sections were evaluated by the Draize scoring system. Erythema and edema were scored as follows: 0 = neither erythema nor edema; 1 = very slight erythema and/or barely perceptible edema; 2 = well-defined erythema and/or slight edema; 3 = moderate to severe erythema or moderate edema, and 4 = severe erythema and/or edema. Thickness of epidermis was measured on digital expanded images of the cross sectioned skin tissue pieces. The samples include 1) control group at 24 h post application (unloaded DDMN patch), 2) control group at 7 days post application (unloaded DDMN patch), 3) treated group at 24 h post application (PRN-loaded DDMN patch) and 4) treated group at 7 days post application (PRN-loaded DDMN patch). There were 12 application sites for each group (3 sites/animal, 4 animal/group). Three measurements were done for each application sites. The total of numbers of measurements were 36 measurements/group.
Difference between epidermal thickness of rats from the unloaded-DDMN-treated group and the PRN-loaded DDMN-treated group was analyzed by unpaired t-test through the GraphPad Prism version 9.1.1 (225) software (GraphPad, USA). Here the p value of < 0.001 was used to indicate significant difference.