Sodium alginate (Cat. No. 7528-1405) with a molecular weight of 120,000–190,000 g/moL was purchased from Daejung Chemicals, Korea. Propylene glycol (99.5% pure; Cat. No. 24300-11000PE) was purchased from Penta Chemicals Limited, Czech Republic. 2-Phenoxyethanol (94% pure; Cat. No. A10786.30) was purchased from Alfa Aesar, United States. 2-Deoxy-D-ribose sugar (Cat. No. 00613) was purchased from Chem-Impex International, United States. Minoxidil (Hair Max^® 2%) was purchased from Sante (Private) Limited, Karachi, Pakistan, and testosterone enanthate (brand name Testoviron Depot^®) was purchased from BAYER Pharmaceuticals, Germany. Autoclaved deionized water was used in the manufacturing process in the current research. All chemicals and reagents were of analytical grade and used without any further purification.

Two different hydrogels, blank-SA and 2dDR-SA, were prepared by simple manual mixing of the constituents in autoclaved sterilized water at RT by using a spatula. The blank-SA hydrogel was composed of 1.4 g of sodium alginate (6.4% w/w), 250 mg of propylene glycol (1.15% w/w), and 82.5 mg of 2-phenoxyethanol (0.377% w/w) as a stabilizer in 20 mL water. The 2dDR-SA hydrogel was composed of 1.4 g sodium alginate (6.416% w/w), 250 mg propylene glycol (1.146% w/w), 82.5 mg of 2-phenoxyethanol (0.375% w/w), and 86.62 mg of 2-deoxy-D-ribose sugar (0.394% w/w) in 20 mL water. The prepared hydrogels (blank-SA and 2dDR-SA) were stored in glass vials at RT.

For FTIR and 2dDR release studies, 10 mL each of blank-SA and 2dDR-SA hydrogels were poured into Petri dishes (100 × 15 mm) and covered with a perforated aluminum foil. These were frozen at −20°C for 20 h and then placed in Labconco’s FreeZone (4.5 L) at −105°C for 24 h. These freeze-dried hydrogels (FD-blank-SA and FD-2dDR-SA) were stored at RT until used for analyses.

The FTIR technique is used to estimate a sample’s interference pattern of emission or absorption. Chemical characteristics of manufactured sodium alginate-based 2dDR hydrogel and sodium alginate Blank-Gel were observed on a spectroscopic analysis (FTIR) system in the frequency range of 4,000–400 cm⁻¹ with 256 sequential scans at 8 cm⁻¹ rulings on a photo audio mode, which is a way to examine materials with no specimen preparation (Thermo Nicolet 6700P, United States spectrometer).

2dDR release from the 2dDR-SA hydrogel was quantitatively determined by Dische’s diphenylamine assay (Jennifer and Mura, 2013). A stock solution of Dische’s reagent was prepared by dissolving 0.75 g diphenylamine in a solution of 50 mL glacial acetic acid supplemented with 750 µL of concentrated sulfuric acid in dark conditions. A fresh solution of 2% v/v ethanol in distilled water was prepared for the assay, and 50 µL of the ethanol solution was added to 10 mL of the diphenylamine solution.

Room-temperature dried 2dDR-SA hydrogel was cut into 2 × 2 cm patches and immersed in PBS in a six-well plate in a sterile environment. The plate was tightly sealed with Parafilm in order to avoid evaporation of PBS and incubated at 37°C and 55% humidity. The release of 2dDR was monitored at different time points (4 h and 1 d, 2 d, 3 d, 4 d, and 7 d). At each time point, the release medium (PBS) from each well was extracted and replaced with fresh media. Dische’s reagent (500 µL) was added to 500 µL of release media and incubated for 10 min at 100°C. Samples were then allowed to cool to room temperature for 10 min before centrifugation at 10,000 rpm for 5 min. The absorbance of the resulting supernatant at 590 nm was measured. The concentration of the released 2dDR was obtained from a calibration curve of known dilutions of 2dDR. Cumulative drug release vs. time was calculated.

Seven-week-old male C57BL/6 mice were purchased from the Center of Excellence in Molecular Biology, Punjab University Lahore, Pakistan. The mice were housed in plastic cages at ambient temperature (25°C ± 2°C) under controlled light/dark cycles of 12 h/12 h and fed standard mouse chow and water ad libitum. They were acclimated to the laboratory environment for 1 week. All the principles of laboratory animal care were followed at the Pre-Clinical Research Facility, IRCBM, CUI, Lahore, and all animal experiments were carried out following the Guidelines for the Care and Use of Laboratory Animals by CUI, Lahore Ethical Committee with Ethical approval certificate number EC/MY/007/22.

In brief, animals were randomly divided into six groups of three mice in each NC and T-1, while four in all other four treatment groups. Treatment groups were referred to as T1–T5, as described in Table 1. The androgenic alopecia model was developed in C57BL/6 mice by intraperitoneally injecting testosterone enanthate at 0.1 mL of 5 mg/mL (20 mg/kg) three times per week for 2 weeks, as previously described in the literature, with slight modifications (Fu et al., 2021). After 2 weeks, hair on the treated back skin (2 × 3 cm) of mice were depilated using a hair removing cream under general anesthesia using ketamine at 80 mg/kg I/P and xylazine at 10 mg@/kg I/P. Approximately, 0.5 mL of each hydrogel (blank-SA and 2dDR-SA) was applied topically on the dorsal side of mice (2 × 3 cm) once daily for 20 days, and the experiment was terminated on day 21. Digital photographs were taken on days 0, 7, 14, and 21 using a DSLR camera. Animals were sacrificed by cervical dislocation before hair and tissue samples were collected.

The treatment groups in experimental animals are given in Table 1.

On day 21, at least 70 hairs per mouse were collected from three mice in each group, as described (Valerie et al., 2001). There are usually four types of hair present in the scalp of C57BL6 mice, which are described as guard, awl, auchene, and zigzag. Large guard hairs are first formed during the embryogenesis of hair. We selected 20 guard hairs from 70 random hair samples. The guard hairs were distinguished mainly by their length and the diameter of the hair shaft or thickness of hair. The length of hair was measured by using a digital vernier caliper, while the hair shaft analysis of guard type hairs was done by taking pictures of the hair shaft portions using an inverted microscope (Optika, Italy) at ×40 magnification.

To evaluate skin morphology and hair regrowth, 2 × 3 cm skin samples were collected on day 21 (after depilation). Samples were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin blocks. Longitudinal and horizontal sections of 5.0-μm-thick skin were prepared and stained with hematoxylin and eosin (H&E). Photographs of H&E-stained sections were taken using an inverted microscope (Optika, Italy). The longitudinal sections were stained using Masson’s trichrome staining to evaluate the area covered by melanin in the hair bulb. The sections were evaluated for follicular length, follicular diameters, and follicular density. The horizontal sections were evaluated for anagen follicle (A) and telogen follicle (T), and the A/T ratio was calculated (Ying et al., 2017) using an inverted microscope (Optika, Italy), and images were captured. Four parameters, 1) number of hair follicles; 2) hair bulb diameter; 3) follicular density (number of hair follicles per mm²); and 4) number of anagen (A) and telogen (T) follicles per millimeter area, were measured to evaluate hair growth using ImageJ software (NIH, MD, United States). To assess the angiogenesis potential of 2dDR-SA hydrogels in promoting hair growth in AGA-induced mice, blood vessels were counted in cross-sections of H&E staining.

All experiments were conducted in triplicate or more, and their results were calculated as mean ± S.D. Statistical analysis (unpaired Student’s t-test) was performed via GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/ttest1.cfm). All results with a p-value ≤ 0.05 were considered statistically significant.

FTIR analysis was used to determine the essential structural and chemical properties of the blank-SA and 2dDR-SA hydrogels and validate the formation of a hydrogel matrix. This method also records structural changes caused by any effects of the drug on the carrier hydrogel. The results of the FTIR spectra of the hydrogels shown in Figure 3A were drawn using Origin software (OriginLab, United States) and cross-checked against literature. The spectra obtained for 2dDR, blank-SA, and 2dDR-SA are presented in Figure 4. Sodium alginate exhibited absorption band characteristics at 3,410 cm⁻¹, which can be due to hydroxyl group (–OH) (Thaned, 2009; Jing et al., 2010); 1,635 cm⁻¹ due to the asymmetric stretching vibration of COO groups; 1,419 cm⁻¹ due to the symmetric stretching vibration of COO groups; 1,050 cm⁻¹ due to the elongation of C–O groups; and 1,294 cm⁻¹ and 1,024 cm⁻¹ due to C–O and C–O–C stretching vibrations of alcoholic hydrogen bonding, respectively (Rúben et al., 2011). Bands at 2,859 cm⁻¹ and 2,970 cm⁻¹ corresponded to C–H stretching, and the bands at 3,410 cm⁻¹ corresponded to the hydroxyl group of propylene glycol (Munirah et al., 2016). Characteristic intense bands associated with O–C, C–C, and C–O stretching modes were observed at 1,078 and 1,042 cm⁻¹. The very intense Raman spectra at 996 and 1,006 cm⁻¹ were the characteristic ring band of 2-phenoxyethanol. The observed intense bands at 750 and 688 cm⁻¹ were assigned to the C–H ring wagging modes of 2-phenoxyethanol (Badawi, 2011). In comparison to the peaks of scaffolds without 2dDR, FTIR spectra show that 5% 2dDR-loaded hydrogel has no peak shift. Thus, 2dDR has no effect on the sodium-alginate structure (Figure 1A).

Developing hydrogels with controlled release of drugs is desirable for wound healing applications. The 2dDR drug release was measured using Dische’s diphenylamine assay. This assay is based on 2dDR being changed by the acidic environment into an aldehyde molecule, which then interacts with diphenylamine to form a blue complex that can be easily measured using UV/Vis absorption spectroscopy. For this purpose, the 2dDR release from room temperature-dried 2dDR-SA was performed in PBS at 37°C for different time intervals (4, 24, 48, 72, 96, and 168 h), and the results are shown in Figure 1B. After 4 h, the 2dDR-SA hydrogel released 70.3% (1.33 mg/2 mL) of the total drug. The release was 79.4% (2.045 mg/2 mL) by day 1; by day 2, 84% release (2.60 mg/2 mL); by day 3, 87% release (2.88 mg/2 mL); and by day 4, 89% release [3.50 mg/2 (Figure1B)]. The 2dDR-SA displayed the same release of 89% (3.50 mg/2 mL) on day 7 as it did on the 4^th day, and this was the release of essentially all of the 2dDR from the hydrogel.

This shows that 2dDR is slowly released at 37°C for up to 96 h, thus reducing the need for repeated treatments on a daily basis.

Hair growth was measured according to the method developed by Fu et al. (2021), by determining the skin color of mice. As melanin accumulated in the mouse skin, the color gradually changed from pink to pinkish white, white, grayish white, gray, and finally dark gray. This is because melanogenic activity of hair follicles (HFs) is tightly correlated with the hair cycle. After 7 days of full anagen phase, the skin retained a dark gray color before turning white once more due to melanocyte death, which denoted the beginning of the catagen phase. This suggests that skin color is a useful indicator of hair regrowth following depilation. The telogenic dorsal skins of C57BL/6 mice were treated topically with blank-SA and 2dDR-SA hydrogel, minoxidil 2% spray, and a combination of minoxidil 2% spray and 2dDR-hydrogel. The hydrogel was applied 5 min after minoxidil spray in the TE-induced mice in order to assess the hair regenerative activities in the AGA-induced androgenic alopecia (AGA) mouse model. It is well known that the dorsal hair of C57BL/6 mice has a time-synchronized hair growth cycle (Figure 2A). The skin color, which is bright pink during the telogen phase and turns gray or black during the anagen phase, is used as an indication that hairs are growing and is recorded in a skin color index (Figure 2C) (Van-Long et al., 2017). Until day 14, the treatment group with TE only (T-1) had extensive patches of skin lacking pigmentation, and the skin was pinkish white, scoring a 2 on the skin color score. By day 21, 30%–40% of shafts were formed in skin areas of T-1, while T-2 containing only the blank-SA hydrogel showed slightly higher skin color scores than T-1, with a skin color score of 2 on day 14 and 4 on day 21 when applied topically, however, the hair growing ability of T-3 (2dDR-SA hydrogel) in the AGA mouse model was significantly higher than that of T-1 and T-2 by day 14. The T3 group expressed a skin color score of 5 and 60%–70% of the dorsum of AGA mice was covered with hairs by day 21, and the dorsum of mice in T-3 was 90% covered with hairs, and the skin color score was 6. By comparing the T-3 group with the NC and T-4 positive control groups, no difference in the skin color score and the appearance of hair shafts at these time points were observed (Figure 2B). Additionally, mice in the T-5 group that were treated by a combination of both 2dDR-SA hydrogel and minoxidil showed white grayish skin by day 14, and hair shafts appeared at day 21. AGA mice in T-5 showed slightly delayed hair growth and change in the skin color score compared to NC, T-3, and T-4, but these parameters are still more promising than those in T-1 and T-2 (Figure 2D).

Guard, awl, auchene, and zigzag hair are four different types of hair found in mouse coats. During embryogenesis, large guard hair develops first, followed by secondary intermediates such as awl, auchene, and zigzag. Awl hair is long and straight; auchene hair has one oblique bend; and zigzag hair (shortest and most frequent) is composed of up to four segments (David et al., 2010). In this section, we only selected guard hair for measurement of the length and diameter of the hair shaft. Twenty guard hairs were plucked from the dorsum of mice, where treatments had been applied. A total of 60 guard hairs from three mice of each group were subjected to analysis. The length of guard hairs was measured by using a digital vernier caliper. The average length measured in NC was 6.04 mm, while the average length from the T-1 TE only group (2.410 mm) was lowest of all the treatment groups, and the T-3 B-SA group showed a slight increase in hair length to 2.63 mm. The average length of guard hair in T-3, T-4, and T-5 as 6.20, 6.19, and 6.02 mm, respectively (Figures 3A, B). On comparing with the normal control, no significant difference was found between T3, T4, and T5, while there was a significant difference between T1 and T2 with NC.

The second-most important factor to assess hair morphogenesis is the thickness of the hairs. We took 20 guard hairs from each mouse from the area where we had applied the treatments on the dorsum. The hair samples were observed at ×4 magnification to measure the thickness of hair shafts. The diameter of hair shafts of 20 guard hairs from all three mice from all six groups was examined. Our analysis showed the highest average hair shaft diameter in the NC group (204 µm) followed by the T-4 (201.2 µm), T-3 (198.60 µm), and T-5 (187.5 µm) groups. The lowest average hair shaft diameter (138.8 µm) was observed in T-1, without any topical treatment, and the diameter of hair shaft in the T-2 group (B-SA) was 152.4 µm. Statistical analysis showed there was no significant difference between T4 and T3, while there was a significant difference between NC and T1, T2, and T5 with the normal control. This clearly shows that 2dDR-SA has affected the morphogenesis of hair by increasing the length and thickness of mouse hair. The hair length and thickness in group T-3 (2dDR-SA) were very well-matched to those of the NC and T-4 positive control groups, which indicated that 2dDR restored the normal morphogenesis of hair by supporting the increase in hair length and thickness (Figures 3C, D).

After 21 days of treatment, H&E- and Masson’s trichrome-stained skin slices were microscopically analyzed, and the following factors responsible for hair growth were examined.