Innovative Hydrogel Formulation Combining Phycocyanin and Probiotic for Enhancing Skin Regeneration and Accelerated Wound Healing: A Preclinical Investigation in Wistar Rats

Abstract

Skin repair is a global concern that requires multifunctional wound healing platforms that accelerate healing and shield the wound from external contamination. Phycocyanin (C-PC) is a natural protein that shows favorable characteristics for skin repair. The present study aims to investigate the synergistic wound healing potential of probiotic Pediococcus pentosaceus and C-PC hydrogel formulation. C-PC whey protein nanofibrils (WPINF) were first prepared and evaluated for particle size, % entrapment efficiency, and zeta potential. Selected WPINF was used to prepare several hydrogel formulations which were further evaluated for physical properties and in vitro C-PC release. Formulation with acceptable physical properties and C-PC release was studied for oxidative stress markers, antioxidant biomarkers, and wound healing effect in animal models. WPINF had acceptable particle size (26.59–153.5 nm), % EE (65.76–95.98%), and zeta potential (22.3–26.1 mV). The developed hydrogels exhibited neutral pH values (6.97–7.10), optimal spreadability (5.60–7.50 cm), and viscosity (123–209 Pa). HEC-based hydrogel (F1) showed superior C-PC release which was found to follow Higuchi diffusion. In vivo, hydrogel formulations (with and without probiotic) showed significantly reduced oxidative stress markers, with MDA reduced by 54.76–98.58% and antioxidant biomarkers (GSH and TAC) ensuring successful delivery of therapeutic compounds. Histological analysis showed improved re-epithelialization, reduced inflammation, and well-formed granulation tissue in treated groups, with probiotic-C-PC group demonstrating additional immunomodulatory effects. The study highlights promising potential of C-PC-probiotic hydrogels as a safe and effective biotherapeutics for wound healing, supporting their advancement into further clinical investigations to validate their efficacy in humans.

Introduction

Recent advancements in natural biotherapeutic approaches for wound healing have demonstrated the efficacy of bioactive compounds from natural sources. These compounds are recognized for their antimicrobial, anti-inflammatory, and tissue-regenerative properties, offering new solutions for chronic and acute wound management. Wound healing is a complicated biological process that includes hemostasis, inflammation, proliferation, and tissue remodeling [1]. The advancement of novel treatment strategies to accelerate wound healing has attracted considerable interest, especially the application of bioactive substances and probiotics. The development of wound healing platforms is essential for both preventing infections and promoting efficient healing. In this context, drug delivery systems are fabricated using hydrophilic biocompatible polymers. Application of this scaffold to wounds significantly accelerated healing, evidenced by enhanced collagen deposition and reepithelialization [2].

Phycocyanin, a pigment-protein complex obtained from the edible cyanobacterium, Spirulina platensis, has emerged as a viable candidate owing to its strong antioxidant, anti-inflammatory, and immunomodulatory activities. Research has shown that phycocyanin can reduce oxidative stress and promote tissue regeneration, rendering it a significant asset in wound management [3, 4]. Concurrently, the potential of probiotics especially lactic acid bacteria in wound healing has been investigated. Probiotics confer advantages by modulating the microbiota, diminishing infection risks, and boosting the immune response. They also generate metabolites, including bacteriocins and organic acids, which can promote tissue repair and protect wounds from bacterial colonization [5].

Natural bioactive macromolecules including C-PC are considered promising alternative for effective wound healing. Phycocyanin has wide range of biological activity including anti-tumor, anti-inflammatory, antioxidant, and wound healing activity. However, this molecule was found to be sensitive to pH, light, as well as temperature which limits its use. For this reason, numerous studies were conducted to protect phycocyanin from degradation. These studies include the use of stabilizing agents or its encapsulation in different types of particles [6]. Whey protein was found to be superior in enhancing C-PC stability in a previous study [7]. This approach offers controlled release of therapeutic agents which is required for extended wound healing process. In addition, encapsulation can offer a solution to many problems encountered by natural bioactive molecules such as limited stability and bioavailability [8].

Biomaterials such as collagen, silk, and bacterial cellulose are gaining traction as components of advanced wound dressings, including hydrogels and nanofibrils. These materials mimic the extracellular matrix (ECM), fostering cellular migration and proliferation. Hydrogels, in particular, have shown promise in maintaining a moist wound environment, reducing pain, and preventing secondary infections [9, 10].

The integration of natural biomolecules into biotherapeutic platforms, coupled with innovations like nanotechnology and targeted drug delivery, highlights a transformative approach in wound care and represents a promising strategy for achieving more effective and sustained wound healing. In this concern, several wound healing platforms have been developed using recent techniques such as bilayer scaffold and 3D printed constructs. These developed techniques demonstrated the ability to promote cell adhesion and migration. Additionally, they could enhance key healing processes, including angiogenesis, collagen synthesis, and epidermal layer formation, all contributing to accelerated wound closure [11].

As clinical trials and preclinical studies continue, these methods aim to offer safer and more effective treatments for diverse wound types [12, 13]. These molecules are known to reduce oxidative stress, regulate inflammation, and promote re-epithelialization. Their antimicrobial effects, particularly against antibiotic-resistant bacteria, also make them ideal for wound healing applications [12, 14]. Similarly, polysaccharides like chitosan and hyaluronic acid provide moisture retention and extracellular matrix (ECM) remodeling benefits, crucial for facilitating tissue repair [15, 16].

Hence, in the present study, C-PC-whey protein nanofibrils were developed as a strategy to promote C-PC stability. The developed nanofibrils were then incorporated with or without a probiotic into gel formulation for subsequent wound healing evaluation in animal models. The incorporation of these bioactive substances could accelerate the healing process by utilizing their synergistic antioxidant, antibacterial, and immunomodulatory properties. The results of this study may provide critical insights into the development of advanced biomaterials for wound care.

Materials and Methods

Materials

Whey protein isolates (WPI), mostly composed of β-lactoglobulin 86% and α-lactalbumin 12%, were sourced from Davisco Foods International, Le Sueur, USA. Hydroxyethyl cellulose (HEC) and carboxymethyl cellulose (CMC) were kindly provided by PHARCO Pharmaceuticals Inc. (Egypt). Carbopol (934) was gift from Delta pharm (Egypt). Triethanolamine (TEA) and Methyl paraben was obtained from El Nasr Chemical Co. (Egypt). Other used chemicals and reagents were of analytical grade. Antioxidant Kits were purchased from Biodiagnostic Company, Cairo, Egypt. The normal human foreskin fibroblast cell lines (BJ-1) were kindly provided by professor Stig Linder, Karolinska Institute, Stockholm, Sweden and maintained in RPMI-1640 medium which was supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 U/mL streptomycin. The cells were grown at 37 °C in a humidified atmosphere of 5% CO₂.

Methods

Preparations of Probiotic

The selected Pediococcus pentosaceus MZ413646 previously isolated and identified via 16 s rRNA sequence [17] was used in this study. It was first isolated from soil sample (Cairo Governorate) and evaluated for probiotic properties and safety attributes. The strain was normally propagated on MRS media at 37 °C in a microaerophilic condition and stored at – 80 °C in 50% glycerol. The culture broth of P. pentosaceus was firstly centrifuged (5000 rpm for 10 min at 4 °C) and washed twice with 0.85% NaCl before incorporation in the pharmaceutical formulation. The strain concentration used in formulation was ranged from 10⁸–10⁹ CFU/mL as indicated by plate counting method.

Preparation of Phycocyanin

The blue-green alga, S. platensis, used in the present study was obtained from the Freshwater Hydrobiology Lab, National Institute of Oceanography and Fisheries (NIOF), Cairo, Egypt. Zarrouk medium was used for culturing S. platensis for 21 days [18, 19]. S. platensis was grown in a batch culture at 32 ± 2 °C, supported by a continuous air pump for aeration, adjusted pH at 9, and light intensity ~ 2000 lx, and a fluorescent lamp for 24-h lighting. The cells were allowed to grow to a density of about 1.0 mg/mL and then harvested by centrifugation.

S. platensis biomass slurry was firstly washed with double distilled water to remove adhered salts and then subjected to 3–10 cycles of freezing (− 20 °C for 30–100 min) and thawing (room temperature for 100 min). The thawed biomass was grounded manually (in the dark) in 10 mL of 0.1 M phosphate buffer solution (pH 6.8) [4], and 1 mL of Tris HCl in the presence of acid-washed neutral sand and then filtered. The mixture was further subjected to freezing (− 20 °C for 30–100 min) and thawing for five freeze–thaw cycles [20], stirred at 150 rpm at 4 °C for 30 min, ultrasonicated ten times at 40 °C with 10-s intervals, centrifuged at 4 °C for 8 min at 7000 rpm, and the blue-colored supernatant was taken for further investigations.

C-phycocyanin calculations were determined using spectrophotometry-based methods on the absorbance ratio. The content of C-phycocyanin was calculated according to [4, 21]. Phycocyanin has a single visible absorbance maximum between 615 and 620 nm.

$$\%C-PC=\left(\frac{{\mathit{OD}}_{620}\times V\times 100}{3.39\times W2\times Dw}\right)$$

where C-PC is the crude phycocyanin (%), A₆₂₀ represents the absorbance of phycocyanin at 620 nm, 3.39 is the extinction coefficient of C-PC at 620 nm, V is the total volume, 100 represents 100%, W2 is the weight of wet biomass, and Dw represents the percentage of dry weight.

The determination of phycocyanin purity is based on the absorbance ratio A₆₂₀/A₂₈₀, and the absorbance at 620 and 280 nm corresponds to phycocyanin and total protein, respectively [22].

$$\text{Purity ratio of C}-\text{PC}=\frac{A_{620}}{A_{280}}$$

Preparation of Whey Protein Isolate Nanofibrils

Following the method described by [23], whey protein isolate was nanofibrillated. Accurately weighed 50 mg of whey protein isolate was dissolved in 1 mL of ultrapure water. A magnetic stirrer was used to agitate the generated solution at 200 rpm for 3 h to ensure complete hydration. The pH of the WPI solution was then decreased to 2.0 by adding 8 M HCl, and the heating procedure was extended for 5 h with moderate stirring at 85 °C; subsequently, the solution beaker was placed in an ice bath for 10 min, effectively stopping the fibrillation process. The whey protein nanofibrils (WPINF) solution was kept at 4 °C for further investigations.

C-PC Encapsulation with WPINF

Using 5 M NaOH, the pH of a completely hydrated WPINF solution containing 50 mg/mL protein was brought down to 7.0. The concentrations of C-PC in solutions were 150, 300, 450, and 600 mg/100 mL (i.e., 3:100, 6:100, 9:100, and 12:100 C-PC to protein ratios). For 12 h, all of the solutions were agitated at room temperature in a dark environment. Then it was homogenized using high-intensity ultrasound with a 40% amplitude for 5 min in an ice bath. A VCX800 (Vibra Cell, Sonics, Newtown, CT, USA) with a 13-mm-diameter probe was used for this procedure. For further analysis, the resultants were stored at – 4 °C and freeze-dried [24].

Polydispersity Index, Mean Droplet Size, and ζ-Potential of the Nanofibrils

Dynamic light scattering (DLS) was used to measure the nanocapsules’ particle size using a Mastersizer 2000 from Malvern Instruments (Malvern, UK). At 25 °C, 3 mL of distilled water was used to dilute 30 µL of the sample. Each sample’s size was represented by the surface-weighted mean diameter (d32), which was determined from the total particle size distribution. A Zetasizer Nano ZS-90 from Malvern Instruments (Worcestershire, UK) was used for particle microelectrophoresis in order to assess the droplet charge (zeta potential) of the nanocapsules.

Gel Formulation

Selected C-PC-loaded nanofibrils were utilized for preparation of hydrogel formulations prepared by various gelling agents (HEC, CMC or Carbopol). Gelling agents were dispersed under continuous stirring (300 rpm) in purified water in which methyl paraben (preservative) was previously dissolved. Hot purified water (50 °C) was used for HEC and CMC and cold water for adopted for Carbopol. TEA was added to Carbopol containing hydrogel under stirring condition to neutralize the Carbopol hydrogel. The required quantity of glycerol was added under stirring condition. Previously developed C-PC loaded nanofibril was then added, and the final weight was completed up to 100 g with water (Table 1). The formulations were then packed in wide mouth containers and placed in a refrigerator for complete hydrogel formation [25, 26].

Table 1.

Hydrogel different formulations with HEC, CMC, and Carbopol

Component (g)	HEC gel (F1)	CMC gel (F2)	Carbopol gel (F3)
CMC	–	1	–
HEC	1	–	–
Carbopol	–	–	0.5
Triethanolamine	–	–	0.5
C-PC equivalent to	0.33	0.33	0.33
Glycerol	10	10	10
Methyl paraben	0.1	0.1	0.1
Water up to	100	100	100

Probiotic containing C-PC gel was developed using the selected formula where probiotic strain was first dispersed in sufficient quantity of double distilled water [27] followed by addition of other gel components as previously described. Oil fraction of Spirulina platensis was incorporated as penetration enhancer.

Visual Examination

The prepared gels were visually examined for color, homogeneity, as well as phase separation.

pH Measurements

The pH value for the developed gels was assessed by a pH meter (MODEL 420, Orion, USA) to confirm non-irritant characters. For pH evaluation, each sample (1 g) was placed in a beaker and allowed to swell with 10 mL. The pH was measured using previously calibrated pH meter. Three measurements were taken at room temperature [28].

Spreadability

Gel formulation (0.5 g) was placed on a glass plate and covered with a second glass plate. A constant weight of (200 g) was placed on the upper plate for 5 min till no further spreading occurs. Diameters of spread circles were noted in cm as spreadability values. Average of three measurement was taken [25, 29, 30].

Viscosity

Viscosity of each gel formulation was measured in Pascal (Pa.) by rheometer (Physica MCR 502, Austria). Each formulation (5 g) was subjected to different shear rates from 1 to 100 s⁻¹. All measurements were performed at 25 °C [31, 32].

Drug Content

The amount of C-PC in 1 g gel preparation was determined spectrophotometrically after dispersing the gel in 10 mL methanol followed by dilution with appropriate amount of phosphate buffer (pH 7.4). Measurements were carried out in triplicate.

Release

The in vitro C-PC release from formulations was performed using dialysis bag method using shaking water bath (Gallent kamp, UK) stirred at 50 rpm and maintained at 37 ± 0.5 °C. Each formulation (2 g) was placed in dialysis bag previously soaked in phosphate buffer (pH 7.4). The dialysis bag was then immersed in 250 mL buffer solution in the shaking water bath. Samples were withdrawn at selected time intervals over a period of 24 h and replaced immediately with fresh buffer. Amount of released C-PC was analyzed spectrophotometrically. Experiment was carried out in triplicate. Release data was fitted to different kinetic models to determine kinetics of C-PC release [33].

Gel Strength

Hydrogel formulation with most promising release profile was evaluated for gel strength by measuring the time needed for 35 g weight to sink 5 cm deep in 50 g gel placed in 100 mL graduated cylinder. Measurement was carried out in triplicate [34].

Fourier Transform Infrared Spectroscopy

Functional groups of phycocyanin were observed by Fourier transform infrared spectroscopy (JASCO FT/IR 4600 FT-IR, Germany). Samples (selected C-PC-nanofibril, and physical mixture of nanofibrils with selected hydrogel ingredients) were ground with spectroscopic grade KBr powder and then pressed into 1 mm pellets for Fourier-transformed infrared (FT-IR) measurement in the frequency range of 4000–400 cm⁻¹ (Mid-infrared region) [35].

Scanning Electron Microscope

The shape/morphology/porosity of the selected hydrogel was observed by scanning electron microscope (SEM). Lyophilized hydrogel samples were mounted on the stub and scanned at different magnifications [36].

SEM was also done to visualize cell adhesion by placing the samples in a 6-well plate. Then, 20,000 normal skin fibroblast cells, BJ-1, with 100 μL culture medium were poured, followed by 48 h incubation at 37 °C and 5% CO₂. The sample was removed, washed with PBS, fixed with 4% glutaraldehyde, and incubated for another 1 h at 4 °C. Finally, the samples were washed with distilled water and increasing concentration of ethanol (starting from 70%) to dehydrate, and then observed under SEM [37].

In Vivo Experimental Design for Evaluation of Wound Healing Potential of CPC-Probiotic Hydrogel

Animals

Fifty six, 2-week-old male Albino Wistar rats (100–120 g) were used. They were maintained under standard laboratory conditions (temperature 25 ± 2 °C), relative humidity 55 ± 5%, and a 12 h/12 h light/dark cycle. All animals were fed with standard pellets and water ad libitum. The experimental animals were given proper care and handling according to the institutional animal ethics committee of the National Research Centre, Egypt (Approval number, 12,050,205), which complies with the guidelines for the care and use of laboratory animals as described by the U.S. National Institutes of Health. Animals were randomly divided into seven groups of eight animals each. Group 1: Negative control group. Groups 2, 3: Negative control rats treated with the C-PC and C-PC-probiotic hydrogel once daily for 10 days (the time of the experiment). Groups 4–7: Animals were subjected to skin wound (2 × 2 cm in size), then the following method was applied: Group 4: served as positive control received no treatment. Group 5: injured rats treated with the C-PC hydrogel once daily for 10 days (the time of the experiment). Group 6: injured rats treated with the C-PC-probiotic hydrogel once daily for 10 days. Group 7: injured rats treated with the standard drug (Mebo cream, an herbal ointment primarily used for wound healing, containing B-Sitosterol, Baicalin, Berberine Beeswax, and Sesame Oil) once daily for 10 consecutive days.

Serum and Tissue Preparation

Blood was collected in clean dry test tube by puncture of the sublingual vein and serum was separated by centrifugation at 4000 rpm for 15 min and kept at – 80 °C. Then, rats were sacrificed under slight diethyl ether anesthesia and the skin wounded tissue rapidly dissected, washed with isotonic saline and dried. The skin wounded tissue was fixed in 10% formalin for histological investigations.

Biochemical Determinations of Antioxidant Parameters

All animal groups were subjected to determine the non-enzymatic, glutathione reduced (GSH), malondialdehyde (MDA), and total antioxidant capacity (TAC), in serum by using standard diagnostic kits according to manufacturer instructions. Glutathione (GSH), total antioxidant capacity (TAC), and malondialdehyde (MDA) levels were estimated colorimetrically by the methods of [38–40], respectively.

Histopathological and Histochemical Examination

Specimens were collected from skin, fixed in neutral buffered formalin 10%, washed, dehydrated, cleared, and embedded in paraffin. Then sectioned at 5 µm thickness and stained with Hematoxylin and Eosin for histopathological examination [41].

Histopathological Lesion Scoring

Wound healing criteria were assessed according to [42]. Re-epithelialization was given a number from 0 to 4. Granulation tissue formation was evaluated from 0 to 4. The degree of inflammation was given 0 to 4 to describe the reduction in inflammatory cells number.

Statistical Analysis

Statistical comparison between groups was performed using SPSS version 9.05 (USA). Significant difference was analyzed by one-way analysis of variance (ANOVA) followed by Co-stat computer program. P ≤ 0.05 was considered significant.

Results and Discussion

Concentration and Purity of C-Phycocyanin

The spirulina biomass was extracted after 21 days of growth. Then, 25 g of wet biomass was dried, yielding approximately 2.5 g dry weight. The C-phycocyanin concentration was 31.02%, C-phycocyanin purity ratio was found to be of food grade (0.704). C-phycocyanin is classified as food grade if A₆₂₀/A₂₈₀ ≤ 0.7, reactive grade if 0.7 < A₆₂₀/A₂₈₀ ≤ 3.9, and analytical grade if A₆₂₀/A₂₈₀ ≥ 4.0 [43]. The previously characterized C-PC was assessed for purity, content, and molecular weight, as well as its biological activity. The results confirmed its potent antioxidant, anti-inflammatory, antiviral, and antitumor properties, supporting the healing effects observed in our study. C-PC has almost no cytotoxic activity towards the human healthy cell line (BJ-1) [4]. The amino acid analysis of C-PC revealed the presence of eight out of nine essential amino acids and eight out of eleven non-essential amino acids, both essential for tissue repair. Additionally, its moderate molecular weight (82.992 kDa) facilitates effective skin penetration.

Formation and Characterization of C-PC-Whey Protein Nanofibrils

C-phycocyanin (C-PC) was encapsulated in nanofibrils formed from whey protein, a well-established and effective strategy for stabilizing and preserving sensitive bioactive compounds [35, 44]. This method leverages the excellent protective properties of whey protein nanofibrils to enhance the stability, bioavailability, and functional integrity of C-PC under various environmental conditions. This method aligns with recent studies in which various biopolymers are used to promote angiogenesis and skin regeneration. For example, Trilayer propolis-sulphated polysaccharide containing scaffold was developed as skin substitute or advanced wound dressing [45].

Table 2 illustrates the essential physicochemical features of whey protein isolate nanofibrils (WPINF) and WPINF infused with different doses of C-phycocyanin (C-PC). The measured parameters included droplet size, polydispersity index (PDI), zeta potential, and encapsulation efficiency. Comprehending these attributes is vital for applications in food technology and nutraceuticals, where stability and encapsulation efficiency are paramount.

Table 2.

Average particle size, surface charge (ζ-potential), polydispersity index (PDI), and encapsulation efficiency of WPINF and WPINF loaded-C-PC with different ratios

Treatment	Droplet size (nm)	Polydispersity index	Zeta potential (mV)	Encapsulation efficiency (%)
WPINF	22.63e ± 7.16	0.231e	− 18.10a ± 5.44	–
WPINF-100 C-PC	26.59d ± 4.78	0.279d	− 22.30b ± 5.25	95.98a ± 1.79
WPINF-150 C-PC	78.45c ± 6.07	0.325c	− 22.50b ± 6.74	92.15b ± 2.15
WPINF-200 C-PC	116.40b ± 8.11	0.612b	− 23.50c ± 8.23	84.65c ± 2.09
WPINF-250 C-PC	153.50a ± 5.06	0.636a	− 26.10d ± 3.92	65.76d ± 2.87

WPINF whey protein isolate nanofibrils, WPINF−100 C-PC 100 mg phycocyanin complexed with 50 mg whey protein isolate nanofibrils, WPINF−150 C-PC 150 mg phycocyanin complexed with 50 mg whey protein isolate nanofibrils, WPINF−200 C-PC 200 mg phycocyanin complexed with 50 mg whey protein isolate nanofibrils, WPINF−250 C-PC 250 mg phycocyanin complexed with 50 mg whey protein isolate nanofibrils

% of change: ¼ Mean of the control group–Mean of the treated group/Mean of the control group x100

% of improvement: ¼ Mean of the treated group–Mean of the diabetic group/Mean of the control group x100

The mean droplet size increases with the concentration of C-PC. WPINF alone exhibits a droplet size of 22.63 nm, whereas the sizes for WPINF complexed with C-PC ranged from 26.59 ± 4.78 to 153.50 ± 5.06, reflecting an increase due to the incorporation of phycocyanin at concentrations from 100 to 250 mg complex based on 50 mg WPINF. This trend suggests that higher concentrations of C-PC result in larger particle sizes, most likely due to aggregation or enhanced interactions between the protein matrix and the phycocyanin. This is consistent with previous studies that showed similar increases in particle size with higher encapsulant concentrations [23, 46, 47].

The PDI values reflect the consistency of the particle size distribution. A PDI below 0.3 typically indicates a tight size distribution, but values over this threshold indicate more variability. WPINF has a PDI of 0.231, which is deemed satisfactory. Nevertheless, when the concentration of C-PC escalates, the PDI concurrently climbs, reaching a value of 0.636 at 600 C-PC. This augmentation may indicate a wider range of particle sizes due to the incorporation of more C-PC into the whey protein isolate nanofibrils, as shown in prior encapsulation research employing whey proteins [48, 49].

The stability of the nanofibrils dispersions can be inferred from the zeta potential values. All treatments have negative zeta potential values, signifying a negative charge and implying effective stability against aggregation owing to electrostatic repulsion. The zeta potential decreases to a more negative value with higher concentrations of C-PC, ranging from − 18.10 mV for WPINF to − 26.10 mV for WPINF-600 C-PC. The rise in negativity may improve stability, consistent with studies highlighting the significance of zeta potential in preserving colloidal stability in food systems [50, 51].

Encapsulation efficiency quantifies the effectiveness of C-PC incorporation into the WPINF matrix. The efficiency diminishes with increasing concentrations of C-PC, peaking at 95.98% for WPINF-150 C-PC and declining to 65.76% for WPINF-600 C-PC. This decrease may indicate that at elevated concentrations, a saturation threshold exists beyond which not all C-PC can be efficiently encapsulated inside the whey protein isolate nanofibrils. This conclusion aligns with recent research demonstrating similar trends in encapsulation efficiency as active component concentrations increase [4, 51].

Gel Formulation and Properties

Visual examination of the fabricated gels showed that the developed hydrogels had soft smooth texture with no signs of grittiness, clumps, or phase separation indicating uniform dispersion of C-PC nanofibrils in the formulated gels. The pH values ranged from 6.97 ± 0.085 to 7.10 ± 0.130 (close to neutral) which is considered suitable for application to skin [52]. Bacterial count was monitored continuously to ensure the viability of lactobacilli in the pharmaceutical formulation and to guarantee the minimum therapeutic level (10⁷ to 10⁹ CFU/mL).

The spreadability is important to ensure uniform application and covering of the wound with drug for optimum healing process [53]. Gel spreading allows uniform application to skin. In addition, spreadability is important for patient compliance. Spreadability was in the range of 5.60 ± 0.20 to 7.50 ± 0.26 indicating ease of application on optimum surface area for C-PC permeation [54]. Similar results were reported in previous studies [25, 55].

The viscosity is considered an essential aspect in semisolid dosage forms which facilitates topical application of the prepared formulation and affects drug diffusion as well as drug release [56]. The viscosity of gel formulations was 122.82 and 209.35 Pa, which is considered suitable for topical applications [57]. Similar results were provided in previous studies [58].

Suitable viscosity and spreadability allow precise application onto the skin. Low viscosity values indicate less stress is required for application and ease in flow from the container (Table 3).

Table 3.

Physical properties of different gel formulations

Gel formula	pH	Spreadability	Viscosity Pa at 100 s−1	Drug content
F1	7.03 ± 0.125	7.50 ± 0.26	122.82 ± 4.85	97.02 ± 1.17
F2	7.10 ± 0.130	6.37 ± 0.15	128.97 ± 2.56	96.87 ± 1.16
F3	6.97 ± 0.085	5.60 ± 0.20	209.35 ± 12.90	97.53 ± 1.76

F1: HEC gel, F2: CMC gel, F3: Carbopol gel

Figure 1 shows the in vitro C-PC release from the prepared gels as well as from the developed C-PC nanofibrils. A slower release was observed for the gels than the nanofibrils due to the effect of gelling agents which act as diffusion barrier by increasing system viscosity [56].

Fig. 1.

(a) In vitro C-PC release from gel formulations. (b) Hydrogel

Slowest C-PC release was observed by Carbopol 934 containing formulation (F3) compared to HEC (F1) and CMC (F2) which was expected by the elevated viscosity of F3 compared to other formulations.

In all formulations, C-PC release was extended to 24 h. Higher release was achieved by HEC as gelling agent (F1). The moderately lower viscosity for this formulation allows C-PC to easily diffuse through gel facilitating drug release [59]. Based on the obtained results, F1 was considered to be superior compared to other hydrogel formulation.

After fitting data to different kinetic models, C-PC release was found to follow Higuchi diffusion (Table 4).

Table 4.

Kinetic analysis of C-PC release from hydrogel formulations

Gel formula	R2
	Zero-order	First-order	Higuchi
C-PC NF	0.968673	0.99831	0.998325
F1 (HEC)	0.929846	0.96332	0.977271
F2 (CMC)	0.966181	0.99037	0.997232
F3 (Carbopol)	0.976457	0.99064	0.991265

The selected hydrogel was evaluated for gel strength to determine the system ability to sustain a gel form. Sufficient gel strength is required to prevent the gel from being easily flushed from application site [55].

The developed gel had gel strength of 37.56 ± 2.92 s which indicates that formulation has sufficient mechanical strength and ability to retain gel state. Gel strength of more than 25 s was reported to be suitable for topical administration since gel strength lower than 25 s will quickly drain away from administration site [60–62].

FT-IR

FT-IR spectral analysis is a rapid tool for identification of encapsulated drugs. FT-IR spectra of C-PC (Fig. 2) showed broad band for N–H and O–H in the range of 3200–3600 cm⁻¹. C-H stretching vibration was observed at 2925.5 cm⁻¹ while peaks at 1640 cm⁻¹ and 1383.67 cm⁻¹ were due to C = O and carboxy stretching vibrations respectively. Similar results were observed in previous findings [63].

Fig. 2.

FTIR spectra of C-PC (C-phycocyanin), Physical mixture of C-PC with hydrogel components (PM), C-PC nanofibril (C-PC NF), and C-PC nanofibril with hydrogel components (NF-PM)

These peaks were observed almost unchanged in its physical mixture of C-PC with hydrogel components (Fig. 2, FM) indicating no alteration of C-PC functional groups suggesting absence of interactions between C-PC and the used polymers.

FT-IR of the developed C-PC NF was consistent with FTIR of C-PC with peak at 1638 cm⁻¹ was reduced and peak at 1386 cm⁻¹ almost disappeared indicating C-PC encapsulation in the fabricated nanoparticles. In the physical mixture of hydrogel components and C-PC-NF (NF-PM), peaks corresponding to C-PC appeared in the same pattern as in C-PC-NF indicating adequate physical compatibility.

Conventional wound dressings, such as bandages, pads, or gauzes, often fail to sustain the moist environment crucial for effective healing [64]. Advancements in biomaterials have led to innovative wound dressings, including hydrogels, nanofibrils, and films. Hydrogels, composed of natural or synthetic polymers through chemical or physical crosslinking, are particularly promising. Their structural similarity to extracellular membranes, bio-adhesive nature, and rheological properties make them well-suited for wound healing. Hydrogels’ hydrophilic nature and high-water content provide a cooling effect that alleviates pain, while their semi-solid consistency offers mechanical strength akin to skin and facilitates diffusion [65].

Unlike creams, ointments, or traditional gauzes, hydrogels conform to complex wound shapes and ensure a moist environment, a critical factor in accelerating wound healing. They absorb wound exudates, maintain optimal oxygen and water permeability, and promote fibroblast proliferation and keratinocyte migration, key processes in tissue repair [66, 67]. Additionally, hydrogels can deliver therapeutic agents over prolonged durations via topical or injectable applications, improving treatment outcomes [68].

The skin possesses a natural ability to regenerate and can typically heal minor injuries through a complex series of biological processes. However, when the extent of damage exceeds the skin’s intrinsic healing capacity, medical treatment becomes necessary. Although considerable progress has been made in the development of wound dressings, achieving effective skin regeneration continues to be a major challenge, primarily due to the ongoing problem of recurrent infections [69, 70]. In wound care, hydrogels perform multiple therapeutic functions—from cooling the wound site and delivering anesthetics in emergency situations to acting as primary dressings that actively support tissue regeneration. They help minimize scarring, reduce inflammatory cell infiltration, promote earlier epithelialization, enhance angiogenesis, and accelerate overall wound healing [71]. Probiotic hydrogels, in particular, have gained recognition for their ability to enhance wound healing through their unique therapeutic properties, especially in chronic wound treatment protocols [72].

SEM for Hydrogel

Surface morphology of the selected hydrogel (F1) was examined under SEM at several magnifications (Fig. 3). Hydrogel appeared porous with rigid texture. The surface was found to be uneven and rough indicating the presence of pores. The porous surface of hydrogel facilitates drug incorporation into the hydrogel network and fluid diffusion throughout hydrogel. In addition, porous nature permits absorption of aqueous fluid into the hydrogel providing additional benefits to wound healing ability of the hydrogel [73, 74].

Fig. 3.

SEM images of hydrogel formulation: (A) magnification 3000 ×, (B) magnification 6000 ×, (C) magnification 8000 ×, (D) magnification 16,000 ×

Cell Adhesion

Figure 4 illustrates the morphology of BJ-1 cells seeded on the selected hydrogel system after 2 days. SEM images indicated favorable cell adhesion where cells appeared as spheres on the fabricated hydrogel. Favorable cell adhesion could be explained by the bioadhesive characters of HEC [75]. The combination of traditional and modern fabrication techniques, such as nanoencapsulation, enables the creation of highly porous biomaterials that promote cell adhesion and support tissue integration, making them ideal for advanced healing applications [2].

Fig. 4.

SEM images illustrating cell adhesion and distribution on the surface of the prepared hydrogel after 48 h of culture. Magnified views reveal cells firmly attached to the hydrogel surface. (A) magnification 500X, (B) magnification 1000X, (C) magnification 2000X, (D) magnification 4000X, (E) magnification 8000X, (F) magnification 16000X

In Vivo Wound Healing Properties of C-PC and C-PC-Probiotic Hydrogels

As compared to normal healthy rats, skin wounded rats showed significant increase in the oxidative stress; MDA by 151.92%, and showed significant decrease in antioxidants biomarkers GSH and TAC by 72.84 and 72.99%, respectively. Upon treating injured animals with C-PC and C-PC-probiotic hydrogels, more or less normalization in the oxidative stress and antioxidants levels was recorded with improvement percentages 54.76 and 98.58% for MDA and improvement percentages 10.13 and 38.22% for GSH and 37.04 and 53.09% for TAC, respectively as compared to the standard drug (87.62, 39.03, and 58.02%, respectively) (Table 5). C-phycocyanin (C-PC), a key biliprotein derived from Spirulina, possesses notable antioxidant and free radical scavenging activities. It functions as a selective cyclooxygenase-2 (COX-2) inhibitor and has been shown to induce apoptosis in RAW 264.7 macrophages stimulated with lipopolysaccharides. Beyond these effects, C-PC is recognized for its potent anti-inflammatory and anticancer properties, further highlighting its therapeutic potential in managing oxidative stress and related pathological conditions [4, 76].

Table 5.

Oxidative stress criteria of the experimental groups

Parameters	Groups
	G1	G2	G3	G4	G5	G6	G7
GSH (nmole/mL)	370.25a ± 23.00	366.55a ± 20.00	350.25a ± 20.00	100d ± 14.00	137.50c ± 14.00	241.50b ± 14.00	244.50b ± 14.00
% change		0.65	3.50	72.99	62.86	34.77	33.96
% of improvement					10.13	38.22	39.03
TAC (mM/L)	0.81a ± 0.11	0.79a ± 0.11	0.75a ± 0.11	0.22d ± 0.02	0.52c ± 0.02	0.65b ± 0.02	0.69b ± 0.02
% change		2.47	7.41	72.84	35.80	19.75	14.81
% of improvement					37.04	53.09	58.02
MDA (nmol/mL)	9.13d ± 0.60	8.8 3d ± 0.60	8.93d ± 0.60	23.00a ± 2.76	18.00b ± 1.76	14.00c ± 2.76	15.00c ± 2.16
% change		3.29	2.19	151.92	97.15	53.34	64.29
% of improvement					54.76	98.58	87.62

G1: Negative control group. G 2, 3: Negative control rats treated with the C-PC and C-PC-probiotic hydrogel for 10 days. G4: positive control (injured, not treated). G5: injured rats treated with the C-PC hydrogel for 10 days. G6: injured rats treated with the C-PC-probiotic hydrogel for 10 days. G7: injured rats treated with the standard drug (mebo cream) for 10 consecutive days

Data are represented by mean ± SD, n = 8 in each group. Statistical analysis is carried out using SPSS computer program, one way analysis combined with post hoc (LSD; least significant variance) (version, 8) coupled with Co-Stat computer program, where different letters between groups are the significance value at P ≤ 0.05

% change = control group-treated group/control group × 100

% of improvement = positive group – treated group/control group × 100

The human skin microbiome plays an essential role in maintaining skin homeostasis by preventing fluid loss, defending against external pathogens through antimicrobial effects, and modulating immune responses [77]. Probiotics have gained significant attention for their ability to enhance skin health and regulate the microbiome, particularly in cases of injured or diseased skin. Probiotics play a crucial role in maintaining a healthy skin microbiome, which is essential for effective wound healing. When incorporated into hydrogel formulations, probiotics offer several benefits that enhance the healing process and support skin regeneration through several mechanisms; (1) microbiome balance and protection as it helping in restoring and maintaining the natural microbial balance of the skin, preventing the overgrowth of harmful bacteria that can cause infections and delay wound healing; (2) antimicrobial properties as most of probiotic strains could produce antimicrobial peptides and organic acids that inhibit pathogenic bacteria, reducing the risk of wound infections [78]; (3) anti-inflammatory effects as it helping in regulating the immune response by modulating inflammatory pathways, reducing excessive inflammation, and promoting a favorable environment for tissue repair [5]; (4) enhanced skin barrier function by reinforcing the skin’s natural defense mechanisms, aiding in moisture retention and protecting against external irritants [72]; (5) stimulation of collagen production as some probiotic strains promote fibroblast activity and collagen synthesis, which accelerates tissue regeneration and wound closure [79]; (6) hydration and pH regulation as it contributes in maintaining optimal skin hydration and pH, creating a more favorable environment for cell proliferation and healing; and (7) biofilm disruption as probiotics can prevent or break down biofilms formed by harmful bacteria, reducing antibiotic resistance and improving the efficacy of antimicrobial treatments [5]. This was confirmed by integrating probiotics into hydrogel-based wound dressings, as formulation with probiotic offered a more biocompatible, infection-resistant, and regenerative approach to wound management, supporting faster recovery while preserving the skin’s natural microbiome.

C-phycocyanin (C-PC), a supramolecular complex of protein and chromophore found in all cyanobacteria, has shown immense therapeutic potential [80]. Recent research highlights its ability to support tissue regeneration, improve wound healing, and exert antioxidant, antimicrobial, and anti-inflammatory effects [81]. For instance, studies indicate that incorporating C-PC into hydrogels enhances antioxidant capacity; reduces inflammatory markers like IL-6, IL-1β, and TNF-α; and accelerates wound closure [82]. Furthermore, C-PC has been shown to mitigate UVB-induced skin damage by preventing collagen degradation, boosting antioxidant enzyme activity, and reducing inflammation, demonstrating its anti-photoaging potential [78]. Additionally, both in vitro and in vivo studies suggest that C-PC can activate pro-apoptotic genes while suppressing anti-apoptotic ones, making it a promising candidate for skin cancer treatment [81].

The application of C-phycocyanin (C-PC) largely depends on its purity, typically assessed by the absorbance ratio A₆₂₀/A₂₈₀, where A₆₂₀ represents the absorbance of C-PC at 620 nm, and A₂₈₀ measures absorbance due to other proteins at 280 nm [3]. A purity level equal to or exceeding 0.7 is adequate for food applications [83]. In this study, the C-PC utilized met the required purity standards for its intended application as topical preparation. However, C-PC’s unique molecular structure poses challenges in developing stable topical formulations, as its stability can be influenced by factors such as temperature, pH, and the choice of excipients during formulation processes [84, 85]. Therefore, C-PC was nanoencapsulated as nanofibrils with whey protein.

Hydrogels, composed of three-dimensional polymer networks with high water content, provide an ideal platform for incorporating hydrophilic substances such as C-PC [86, 87]. When a hydrogel is applied to the skin, the active compound must first be released from the formulation and then penetrate the outermost layer of the skin, the stratum corneum. This layer acts as the primary barrier and rate-limiting step for drug delivery. While the skin poses a challenge as a protective barrier, it also offers a significant advantage due to its large surface area for drug application [88, 89].

For successful passive absorption and penetration through the stratum corneum, the drug must meet specific physicochemical criteria, including a molecular weight below 500 kDa, moderate lipophilicity, and adequate solubility in both aqueous and lipid phases [90]. C-PC’s hydrophilic nature and high molecular weight present unique challenges for its inclusion in topical formulations. Addressing these issues, enhancing skin permeability remains a critical area of focus in pharmaceutical research [91, 92]. The use of penetration enhancers in topical systems has proven effective in overcoming these skin barrier properties. These enhancers disrupt the skin’s diffusional pathways, increasing permeability and facilitating drug delivery across the epidermis [93, 94]. For this reason, the lipid fraction of Spirulina platensis was used in hydrogel preparation as a penetration enhancer.

Histopathological Findings and Lesion Scoring

The skin, part of the integumentary system, is the body’s largest and primary protective organ, serving as a physical barrier against external elements and pathogenic microorganisms. As the first line of defense, the skin is vulnerable to damage from thermal injury or trauma [95]. Following an injury, initial colonization by the skin’s native microbiota, including Staphylococcus aureus and Streptococcus pyogenes, typically occurs. Soon after, gut flora from the individual can also colonize the wound, further complicating its microbial environment [96].

The increasing resistance of pathogens to systemic and topical antibiotics poses challenges in managing wounds and infections. This has shifted attention toward alternative therapies such as topical probiotics, which are gaining recognition for their potential in wound treatment. The imbalance or absence of skin microbiota significantly affects both the pace and quality of wound healing [97].

Several studies have demonstrated that topical probiotics offer promising outcomes in wound care. These live microorganisms exhibit antibacterial and antimicrobial effects, regulate inflammation, and enhance infection control, thereby improving the wound healing process [5]. Probiotics have been proposed as an alternative to antibiotics for treating infections. Research in animal models has shown their efficacy in managing surgical wounds and burn injuries. Specifically, probiotics have been reported to act against methicillin-resistant Staphylococcus aureus (MRSA) and as antimicrobial agents that accelerate the wound healing process [97, 98].

Histological H&E-stained sections of rat skin (Figs. 5 and 6) showed different responses to treatment. G1 (control rats) showed normal histological structure of epidermal and dermal layers. G2 (control rats treated with C-PC hydrogel) showed normal histological structure of epidermis and dermis indicating no allergic reactions, sensation, irritation, or any other side effects. G3 (control rats treated with C-PC-probiotic hydrogel) showed normal histological structure of epidermis and dermis indicating also no side effects. G4 (skin wounded rats) showed irregular organized tissue with angiogenesis, heavy inflammatory cells infiltration in dermal layer, intense tissue necrosis, and hemorrhage at wound area. G5 (skin wounded rats treated with C-PC hydrogel) showed re-epithelization with well-formed blood capillaries, re-epithelization, and hyperplasia of epidermal layer and well-formed granulation tissue at wound area. G6 (skin wounded rats treated with C-PC-probiotic hydrogel) showed haphazardly arranged organized tissue, moderate inflammatory cells infiltration and re-epithelization with thin epidermal layer. G7 (skin wounded rats treated with the reference drug, mebo) showed well-formed organized granulation tissue and re-epithelization.

Fig. 5.

Histological H&E stained sections of rat skin (× 100). G1: Control rat skin showing normal histological structure of epidermis (ED), dermis (D), keratin layer (Kr), hair follicles (HF), and glands (G); G2: Control rat skin treated with C-PC hydrogel showing normal histological structure of epidermis and dermis; G3: Control rat skin treated with C-PC-probiotic hydrogel showing normal histological structure of epidermis and dermis; G4: Skin wounded rat showing irregular organized tissue (IT) with angiogenesis (Ang), heavy inflammatory cells infiltration (IC) in dermal layer, intense tissue necrosis and hemorrhage (H) at wound area, and scab (Sc) formed of necrotic tissue covering the wound; G5: Skin wounded rat treated with C-PC hydrogel showing re-epithelization (RE) with well-formed blood capillaries (Bc), re-epithelization and hyperplasia (HP) of epidermal layer and well-formed granulation tissue (GT) at wound area; G6: Skin wounded rat treated with C-PC-probiotic hydrogel showing haphazardly arranged organized tissue, and re-epithelization with thin epidermal layer; G7: Skin wounded rat treated with the reference drug showing well-formed organized granulation tissue and re-epithelization

Fig. 6.

Histological H&E stained sections of skin wounded rat (× 400). G4: skin wounded rat showing tissue necrosis (N) at wound area and inflammatory cells infiltration (IC) in dermal layer; G5: skin wounded rat treated with C-PC hydrogel showing few inflammatory cells infiltration in dermal layer and well-formed granulation tissue (GT) at wound area; G6: skin wounded rat treated with C-PC-probiotic hydrogel showing few inflammatory cells infiltration in dermal layer with notable granulation tissue formation; G7: Skin wounded rat treated with the reference drug showing moderate inflammatory cells infiltration and well-formed granulation tissue

Table 6 illustrated histopathological lesion score of different groups: re-epithelialization was given a number from 0 to 4. Granulation tissue formation was evaluated from 0 to 4. Reduction in inflammation was given from 0 to 4. The results revealed the superiority of C-PC-probiotic hydrogel in alleviating wound and accelerating its healing compared to the reference drug.

Table 6.

Wound healing criteria of the experimental groups

Criteria	G4	G5	G6	G7
Re-epithelialization score	0	2	4	3
Granulation tissue formation	1	2	3	3
Inflammation score	0	2	3	4

G4: positive control (injured, not treated). G5: injured rats treated with the C-PC hydrogel for 10 days. G6: injured rats treated with the C-PC-probiotic hydrogel for 10 days. G7: injured rats treated with the standard drug (mebo cream) for 10 consecutive days. 0: no lesion, 1: low lesion (≤ 10%), 2: medium lesion (≤ 30%), 3, 4: high lesion (≥ 60–70%)

In a preclinical investigation, Satish et al. [99] demonstrated that a single application of Lactobacillus plantarum (3 × 10⁸ CFU, ATCC 10241 strain cultivated in MRS broth) reduced Pseudomonas aeruginosa colonization by approximately 37% on thermal burn wounds (100 °C) induced on the dorsal skin of male Dutch Belted rabbits [99]. This probiotic intervention significantly reduced the injury-induced accumulation of type I collagen mRNA by around 50%, leading to a lower overall total collagen protein level compared to untreated wounds and healthy skin. Remarkably, Lactobacillus plantarum appeared to influence the collagen arrangement post-injury, shifting from mature type I collagen to the more regenerative type III collagen, which is preferentially associated with effective wound healing and skin repair.

Additionally, in agreement with our study, encapsulated C-PC showed enhanced therapeutic outcomes, including sustained release, improved antioxidant activity, and superior wound-healing efficiency as proved with previous in vivo studies that revealed accelerated wound closure with no signs of irritation or allergic responses, indicating its excellent biocompatibility and promising therapeutic potential [100].

The clinical signs of the wound at the end of the experiment (Table 7) proved the superiority of the probiotic-C-PC hydrogel formulation which led to complete closure of the wound compared to other treatments including the reference drug. These results underscore the promise of C-PC-probiotic hydrogels as innovative and safe biotherapeutic solutions for enhancing wound healing, paving the way for deeper clinical exploration.

Table 7.

Clinical signs of the wound at the end of the experiment

G4: positive control (injured, not treated). G5: injured rats treated with the C-PC hydrogel for 10 days. G6: injured rats treated with the C-PC-probiotic hydrogel for 10 days. G7: injured rats treated with the standard drug (mebo cream) for 10 consecutive days

Conclusion

The study establishes that C-PC and C-PC-probiotic hydrogels are safe and effective for wound management, with the probiotic-enhanced formulation showing additional benefits in modulating oxidative stress and immune response. Moreover, the integration of probiotics into C-PC-based hydrogels represents a novel approach that leverages the synergistic effects of natural bioactives and beneficial microorganisms. This combination not only enhances wound healing through antioxidant and anti-inflammatory mechanisms but also supports microbiome balance at the wound site, potentially reducing infection risks and promoting tissue regeneration. The biodegradable and biocompatible nature of these hydrogels aligns with the growing demand for eco-friendly and minimally invasive medical therapies. These findings suggest a potential clinical application for biotherapeutic hydrogels in treating chronic and acute wounds, paving the way for natural, sustainable, and patient-friendly wound care solutions. Further studies could explore scaling production, evaluating long-term effects, and conducting human trials to validate these promising results.

Authors’ Contributions

A.N: Conceptualization, methodology, formal analysis, writing original draft, visualization, funding acquisition, writing – review & editing. T. N. S.: Methodology, writing. A. E.: Methodology, writing. M. A. A.: Conceptualization, Methodology, formal analysis, writing original draft, writing – review & editing. H. F. A.: Methodology, writing. E. A. Y.: Methodology, writing. N. S. F.: Conceptualization, methodology, writing, funding acquisition. All authors reviewed the manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Open access funding was provided by the Science, Technology & Innovation Funding Authority (STDF) in collaboration with the Egyptian Knowledge Bank (EKB) under the Springer Nature agreement.

Data Availability

All data and materials used in this research work were included and available in the manuscript.

Declarations

Ethics Approval

The study was conducted in accordance with the ethical procedures and policies approved by Animal Care and Use Committee of National Research Centre, Egypt, which complies with the guidelines for the care and use of laboratory animals as described by the U.S. National Institutes of Health. Approval Number 12050205.

Consent to Participate

Not applicable.

Consent for Publication

All authors consent to publication.

Competing interests

The authors declare no competing interests.