1. INTRODUCTION:

Chronic wound management presents a formidable challenge in modern healthcare, particularly for populations grappling with age-related complications and metabolic disorders like diabetes. The intricate interplay between impaired glucose metabolism and compromised tissue repair mechanisms in diabetic patients often results in persistent, non-healing wounds that defy conventional treatment approaches^1,2. This complex pathophysiology necessitates innovative therapeutic strategies that can address the multifaceted nature of diabetic wound healing.

The limitations of systemic drug administration in treating diabetic foot ulcers (DFUs) have spurred interest in localized delivery methods^3-5. Topical drug delivery emerges as a promising alternative, offering the potential to concentrate therapeutic agents at the wound site while minimizing systemic exposure and associated side effects^6,7. This targeted approach aligns with the growing emphasis on precision medicine, aiming to maximize therapeutic efficacy while reducing overall drug burden^8-10.

However, the path to effective topical delivery is fraught with challenges. The hostile microenvironment of chronic wounds, characterized by proteolytic enzymes and fluctuating pH levels, can rapidly degrade therapeutic compounds^11-13. Moreover, the dynamic nature of wound exudates can lead to unpredictable drug release profiles and premature clearance from the target site. These obstacles, compounded by the rising specter of antibiotic resistance, underscore the need for sophisticated drug delivery systems that can withstand the harsh wound milieu while maintaining therapeutic efficacy.

Nanotechnology offers a paradigm shift in addressing these challenges. By leveraging the unique physicochemical properties of materials at the nanoscale, researchers can engineer drug delivery vehicles that interact with biological systems in ways previously thought impossible^14-16. Solid lipid nanoparticles (SLNs) represent a particularly promising class of nanocarriers for wound healing applications. Their lipidic nature confers biocompatibility and biodegradability, crucial attributes for topical applications where tissue irritation must be minimized¹⁷. The versatility of SLNs in encapsulating a wide range of therapeutic agents, from small molecules to macromolecules, opens up new possibilities for combination therapies tailored to the complex needs of diabetic wounds¹⁴.

The incorporation of Rutin (RN) into SLNs marks a significant advancement in this field. Rutin, a bioflavonoid with a remarkable array of biological activities, holds immense potential in wound healing. Its anti-inflammatory and antioxidant properties address two critical aspects of chronic wound pathology: persistent inflammation and oxidative stress. By modulating these processes, Rutin-loaded SLNs (RN-SLNs) could potentially break the cycle of chronic inflammation that impedes healing in diabetic wounds¹⁸.

Furthermore, Rutin's ability to stimulate collagen synthesis and enhance vascular integrity directly addresses the structural and perfusion deficits characteristic of diabetic ulcers. The potential antibacterial effects of Rutin add another layer of therapeutic benefit, particularly relevant in the context of infection-prone diabetic wounds¹⁹. However, it's crucial to note that while these properties are promising, their translation into clinical efficacy requires rigorous evaluation.

In our current work, we have effectively developed and assessed the therapeutic effectiveness of a lipid-based drug delivery system utilizing the bioactive chemical Rutin (RN) for the goal of efficiently treating diabetic ulcers. In our earlier research, we successfully produced and statistically optimized the solid lipid nanoparticles (RN-SLNs). In addition, this study evaluated the effectiveness of RN-SLNs by measuring several in vivo parameters to determine the performance of the drug delivery carrier, thereby assuring the effective treatment of diabetic wounds.

2. MATERIALS:

Streptozotocin was procured from Merck Chemicals, Germany. Gluco One BG-03 50 Strip, hair removal cream, gloves, paper towels, surgical forceps, and scissors were sourced from a local pharmacy. The procurement of Carbopol 934, triethanolamine, phosphate buffer of pH 7.4, and bacteriologically graded methanol (for research purposes) was facilitated through Hi-media, India. Ultrapure water from the Milli-Q water system was utilized for the preparation of aqueous and buffer solutions. All additional reagents employed were of analytical grade.

2.1. Animals:

Male Sprague Dawley (MSD) rats weighing between 150-200 gm were employed for the experiments. The animals were housed under standardized conditions with ad libitum access to food and water²⁰. The photoperiod was maintained at 12 hours of light and 12 hours of darkness. All animal experimentation strictly adhered to the protocol sanctioned by the Institutional Ethical Committee of Pinnacle Biomedical Research Institute (PBRI), Bhopal (M.P.), in accordance with CPCSEA guidelines, India.

2.2. Experimental Protocol:

Following the formulation, optimization, and characterization via in-vitro studies, bioactive-loaded lipid carriers, specifically RN-SLNs, were selected based on in vitro results and subsequently integrated into separate gel formulations for further in vivo experimentation²¹. The pre-fabricated RN gel and RN-SLNs gel formulations underwent in vivo studies to ascertain their behavior within the biological milieu.

3. METHODOLOGY:

3.1. Preparation of RN-SLNs gel:

Nanoencapsulation of pharmaceuticals represents a frontier in drug delivery, with solid lipid nanoparticles (SLNs) emerging as a promising vehicle. The integration of Rutin (RN) into SLNs exemplifies the potential for enhancing bioavailability and targeted delivery of antidiabetic medications. This innovative approach merges the fields of nanotechnology and pharmacology, potentially revolutionizing diabetes management. The synthesis of RN-loaded SLNs (RN-SLNs) employs a sophisticated dual-step methodology, combining hot homogenization with ultrasonication^22-24. This process begins with the careful selection of Precirol ATO 5 as the lipid matrix, chosen for its biocompatibility and ability to form stable nanostructures. The dissolution of RN within this lipid phase marks a critical step, as it directly influences drug loading efficiency and release kinetics. A binary solvent system comprising methanol and acetone in equal proportions facilitates the complete solubilization of the drug-lipid mixture^22-25. This strategic choice of solvents optimizes the incorporation of RN into the lipid matrix, potentially enhancing drug entrapment efficiency. The aqueous phase, pivotal for nanoparticle formation, incorporates Poloxamer 188 and Phosphatidylcholine as surfactant and cosurfactant, respectively. This combination not only stabilizes the nanoparticles but also modulates their surface properties, potentially influencing cellular uptake and biodistribution. The thermal manipulation of both phases to 80°C prior to homogenization is crucial. This elevated temperature ensures the lipid remains in a molten state, facilitating uniform drug distribution and nanoparticle formation. The subsequent high-speed homogenization at 30,000 rpm for 10 minutes represents a critical step in achieving nanoscale dimensions and uniform particle size distribution. Following homogenization, the application of probe sonication for 5 minutes further refines the nanoparticle structure^26,27. This step is instrumental in reducing particle size and enhancing size uniformity, factors that significantly impact the pharmacokinetic profile of the encapsulated drug. The controlled cooling to room temperature and subsequent dilution with deionized water is essential for solidifying the lipid matrix and stabilizing the nanoparticle dispersion^26,27. For application over wounded skin RN-SLNs were dispersed in 0.5% w/v Carbopol gel.

3.2. In vitro Characterization of gel:

3.2.1. Determination of pH value:

The pH values of the RN-SLNs loaded biogel formulations were evaluated at room temperature employing a benchtop digital pH meter. Each measurement was recorded three times and averaged²⁸.

3.2.2. Drug Content Determination:

To determine the drug content, a specific amount of RN-formulated gel (1g) was dissolved in 100mL of methanol. The gel solution in the volumetric flask underwent mechanical shaking for 2hours to ensure drug solubilization. Subsequently, the solution was filtered through a 0.45µm membrane filter. After an appropriate dilution, HPLC measurements were conducted²⁹.

3.2.3. Viscosity study:

Viscosity studies of the developed biogel formulations were conducted using a Brookfield digital viscometer (Model RV-1) equipped with spindle No. 3, all under room temperature conditions³⁰. The formulated nanogel was introduced into the small adapter of the Brookfield synchro-electric viscometer.

3.2.4. Spreadability:

The spreadability analysis of the biogel formulation (RN-SLNs) was carried out utilizing a modified parallel plate method. A 100mg sample was applied onto the lower plate of the device to achieve an even layer of the formulation. Subsequently, a mass of 5g was placed onto the pan, which was previously attached to the upper slide. The time in seconds required for the upper plate to traverse the length of the lower plate was recorded. This time served as an indicator of spreadability, reflecting the ease with which the formulation could spread on the skin³¹.

The spreadability was then quantified employing the following formula:

3.3. Skin irritation study:

The skin irritation potential of the formulated RN-SLNs gel was assessed using MSD rats. In preparation for the study, the hair on the dorsal area of the rats was trimmed using a 1mm hair clipper (Sterling). This hair-free region was cleansed with a moistened cotton swab, and the formulations, namely RN-gel and RN-SLNs gel, were topically applied to the designated area32,33. Subsequently, the skin was monitored for any discernible changes, such as erythema (redness), at 24-, 48-, and 72-hours post-application. The mean erythema scores were recorded on a scale from 0 to 4, based on the degree of erythema observed:

· No sign of erythema = 0,

· Slight erythema (barely seeming-light pink) = 1,

· Moderate erythema (dusky pink) = 2,

· Moderate to severe erythema (bright red) = 3,

· Severe erythema (extreme redness) = 4

3.4. Ex-vivo skin permeation and retention study:

Ex-vivo studies on drug permeation and skin retention of gel formulations (RN-SLNs) were conducted using shaved, excised dorsal skin from MSD rats, devoid of adherent fat layers, in static Franz diffusion cells (PermeGear Inc. USA) with a contact surface area of 0.64 cm². For ex-vivo drug permeation and skin retention of the delivered drug via gel formulations, fresh dorsal skin of rats was employed. Subcutaneous fat was meticulously removed from the skin using dampened forceps and subsequently washed multiple times with PBS of pH 5.8. The skin was then punched with a diameter of 15mm and positioned between the donor and receptor compartments, with the stratum corneum facing the donor^34,35. The formulation, containing approximately 10mg of the RN, was introduced into the donor compartment. A release medium consisting of PBS of pH 5.8 with methanol (30:70v/v) was employed to maintain a sink condition. The temperature of the medium was stabilized at 36±2°C to simulate the body temperature of rats and stirred at 250rpm. At specified intervals (0, 1, 2, 3, 4, 5, 6, 8, 12, and 24hours), samples were withdrawn from the receptor compartment and replaced with an equivalent volume of fresh medium. These samples were analyzed for skin penetration using a developed HPLC method³⁶.

Subsequently, for the drug retention study, the skin was detached from the diffusion cell after 24hours of skin permeation study. Any residual gel on the outer surface of the excised skin was thoroughly washed out with 10 ml of water. Afterward, the drug in the residual gel was extracted by adding 10ml of methanol to the rinsed sample. The layers of skin (epidermis and dermis) were then carefully separated using an electric dermatome and stored at -20°C, wrapped in aluminum foil until needed. The skin layers were subsequently homogenized separately with 5ml of release medium, and the drug was extracted by adding 5ml of methanol to all samples. These samples underwent centrifugation (for 15 minutes at 10000RPM), and the supernatants were analyzed using the developed HPLC method³⁷. The extracted drug from the skin was quantified for drug retention. These procedures were replicated in triplicate.

3.5. Wound Healing Activity on STZ-induced Diabetic Animal Model:

3.5.1. Induction of Diabetes:

Diabetes Mellitus (DM) was chemically induced in MSD rats using the STZ induction method. Briefly, after an overnight fast, a single intraperitoneal injection of streptozotocin (STZ) (65mg/kg) freshly prepared in 0.1 M sodium citrate buffer (pH 4.5) was administered (Table 1). Glucose levels were measured on the 8th day after STZ administration using a glucometer³⁸. Rats with fasting blood glucose levels exceeding 225mg/dL were considered diabetic. Throughout the study, water intake and weight were monitored. To confirm the diabetic state, fasting blood glucose levels were rechecked on the day of wound creation. The detailed protocol is outlined in the following scheme.

3.5.2. Wound Generation:

Following diabetes induction, rats from each group were anesthetized with ketamine (5mg/kg body weight), and the hair on their dorsal surface was shaved. Subsequently, an excision wound was created by cutting a 100 mm² full-thickness circular area on the upper back of each rat in every group^39,40. These wounds were left uncovered in an open environment. The prepared hydrogels, RN-gel, and RN-SLNs-gel were topically applied once a day until complete epithelization. This model encompassed various wound healing evaluation parameters including wound contraction and epithelization time.

3.5.3. Wound Contraction:

The borders of the wounds were outlined on transparent paper using a fine-tip permanent marker on the day of wounding. The area (mm²) within the outlined borders of each tracing was determined planimetrically⁴¹. The wound area for each animal was measured at predetermined time intervals, commencing at 3hours post-wounding, and subsequent measurements were taken on days 4, 8, and 14 post-wounding. Changes in wound area were monitored regularly, and the rate of wound contraction was calculated using the formula:

Zero day wound area – Unhealed wound

Wound (%) = ---------------------------------------- x 100

Contraction Zero day wound area

3.5.4 Epithelization time:

Epithelization time represents the duration required for the development of new epithelial tissue to cover the wound site⁴². It was calculated as the time taken for complete removal of scar tissue, leaving no wound behind.

3.6. Statistical Analysis:

Statistical analysis was performed using OriginPro (trial version, 2023). The significance of the obtained statistics was assessed through one-way ANOVA followed by Bonferroni’s test, with a probability threshold of p<0.05 indicating statistical significance.

4. RESULT AND DISCUSSION:

4.1. In vitro Characterization of gel:

4.1.1. pH of topical gel formulation:

The pH level of a topical gel is a pivotal factor influencing its skin compatibility and therapeutic effectiveness. In the case of the RN-SLNs gel formulation, the recorded pH was 6.5±1.05. This nearly neutral pH aligns seamlessly with the skin's natural pH, which not only enhances skin tolerance but also minimizes the likelihood of irritation⁴³. This optimal pH ensures the formulation's suitability for dermatological application, prioritizing patient comfort without compromising efficacy.

4.1.2. Drug Content Determination:

Accurately determining drug content is a fundamental step in pharmaceutical formulation. In our investigation, the RN-SLNs gel formulation exhibited an impressive drug content of 95.77±2.51% for RN, assessed through HPLC analysis. This high drug content underscores the efficient encapsulation of RN within the SLNs. It affirms that a significant proportion of the loaded drug is effectively retained within the gel, thereby ensuring the formulation's therapeutic effectiveness^44,45. The precise quantification of drug content holds paramount importance for consistent dosing and efficacy, and our findings affirm the reliability of this gel in delivering the intended drug dosage.

4.1.3. Rheological and Spreadability Study:

The exploration of rheology, delving into the flow and deformation characteristics of materials, holds pivotal importance in comprehending the physical attributes of gel formulations⁴⁶. In our research, we meticulously scrutinized the viscosity and spreadability of the biogel laden with RN-SLNs. Viscosity serves as a gauge of the gel's propensity to resist flow, and our observations revealed a discernible reduction in viscosity with escalating shear rates (refer to Figure 1). This characteristic proves advantageous for a topical gel, signifying that the formulation can be smoothly applied to the skin, thereby enhancing contact and absorption potential.

Figure 1. Viscosity behavior of formulated biogel.

Spreadability stands as a pivotal attribute, intertwining patient compliance with therapeutic effectiveness. Our findings reveal commendable spreadability values for both the plain gel (6.1±2.14g.cm/sec) and the RN-SLNs gel (6.4±1.13g.cm/sec). This attests to the seamless and comfortable application of these gel formulations onto the skin, assuring an effective distribution of the drug at the application site. Such optimal spreadability plays a paramount role in augmenting patient comfort and guaranteeing precise drug delivery to the intended skin area.

4.2. Skin irritation study:

The outcomes reveal marked skin irritation caused by the RN-gel, whereas RN-SLNs gel exhibited only slight to very slight irritation, indicated by mean erythema scores. Visual assessments demonstrated the least irritation with the bioactive encapsulated in the lipid carrier combined with the gel, ensuring accurate and controlled drug delivery to the wounds⁴⁷. This contrasted starkly with plain RN-gel formulations where the entire bioactive was in direct contact with the skin and wound (Figure 2). Ideally, a novel formulation should induce minimal to no irritation, aligning with prior studies. Hence, the formulated gel can be deemed safe for topical application.

Figure 2. Skin irritation study of prepared gel formulation. A. Control. B. RN-gel and C. RN-SLNs gel.

4.3. Ex-vivo skin permeation and retention study:

Ex-vivo studies on drug penetration and skin distribution were conducted using rat dorsal skin, with results depicted in Figure 3. The bioactive content in the epidermis (In RN-gel, RN= 10.14µ/cm²; In RN-SLNs gel, RN= 19.34µ/cm²) exceeded that in the dermis (In RN-gel, RN= 8.05µ/cm²; In RN-SLNs gel, RN= 15.11 µ/cm²). Drug retention was evident in both the epidermis and dermis, the sites of drug activity. Notably, significantly less RN was detected in the receptor compartment with the RN-SLNs gel formulation, signifying limited systemic circulation, compared to RN-gel. Furthermore, the skin distribution study revealed that RN-SLNs gel formulations efficiently delivered the drug with restricted systemic circulation, presenting themselves as promising carriers for diabetes-related wound treatment. It was observed that the enhanced solubility of the drug in the lipid lattice facilitated RN delivery to the skin surface^48,49. This, in turn, resulted in a substantial concentration gradient, particularly at the epidermis. It is stated in the literature that the lipid-rich environment of the epidermis permits lipophilic compounds like RN to act as drug reservoirs in the intercellular space of epidermal cells. Following drug release from the lipid carrier-based gel, RN primarily accumulates in the epidermis (topical delivery) due to the heightened hydrophilicity of the dermis, limiting significant partitioning of the hydrophobic drug. Additionally, wounded skin exhibits higher permeability than normal skin^50,51. Therefore, in animal skin, a greater deposition of the drug in the epidermis is anticipated, rendering these lipid carriers more effective.

Figure 3. Ex-vivo drug distribution in rat skin.

4.4. Wound Healing Activity:

4.4.1 Induction of Diabetes:

On the 8th day after STZ administration, blood glucose levels were assessed, confirming diabetes induction across all animal groups⁵². The results are detailed in Table 2. Notably, Group II and IV exhibited a slight reduction in blood glucose levels on the 14th post-wounding day compared to Group-I and Group III. This observation may be attributed to the presence of RN in SLNs, corroborating earlier reports on the anti-diabetic properties of RN.

Table 2. Blood glucose level (mg/dl) in each animal group at different times.

Group	Blood glucose level (mg/dl)
Group	0^th Day	8^th Day of STZ administration	21^st day of STZ administration
Group I	109.10±27.16	311.23±18.19	317.01±25.18
Group II	121.17±21.56	404.10±41.06	314.78±18.06
Group III	114.60±21.70	297.10±25.70	221.08±25.10
Group IV	99.62±23.62	256.52±30.09	223.61±22.01

4.4.2. Wound Contraction and Epithelization Time:

Using a fine-tip permanent marker, wound borders were meticulously outlined on transparent paper, and their areas in square millimeters were determined planimetrically⁵³. Measurements were taken on day 0, beginning at 3hours post-wounding, and subsequently on days 4, 8, and 14. The results were expressed as a percentage of wound contraction (Figure 4).

Figure 4. Percent wound contraction in various experiment groups.

The findings indicate a time-dependent increase in wound area reduction across all animal groups, as detailed in Table 3. Notably, Group IV, treated with RN-SLNs, exhibited the highest wound closure on the 14th day post-wounding, followed by Group II (Plermin Gel) and Group III (RN-gel). The control group exhibited limited healing on the 14th day post-wounding. Notably, in all experimental groups, Group III (treated with RN-gel) showed less wound closure compared to the standard treatment group.

Epithelization periods were also documented in Table 3, revealing significant results in both treated and control groups. No toxicity to epidermal skin cells was observed in any treated group⁵⁴. The groups treated with RN-SLNs-gel and Plermin Gel exhibited enhanced restoration of normal skin architecture, achieving complete epithelization within 19 days (Figure 5). RN played a pivotal role in expediting wound closure and facilitating rapid epithelization in the treated group, surpassing both control and standard groups.

Figure 5. Representation of wound contraction after treatment

5. CONCLUSION:

The development and evaluation of Rutin-loaded Solid Lipid Nanoparticles (RN-SLNs) represent a significant advancement in the field of diabetic wound management. This innovative approach addresses the multifaceted challenges of chronic wound healing in diabetic patients, offering a promising alternative to conventional treatment modalities. The successful synthesis and optimization of RN-SLNs demonstrate the feasibility of encapsulating bioactive compounds within lipid-based nanocarriers for targeted delivery to wound sites. The careful selection of materials and processing techniques resulted in a stable, biocompatible formulation capable of protecting the encapsulated Rutin while facilitating its controlled release. In vivo assessment of RN-SLNs revealed their potential to modulate key aspects of the wound healing process. The nanoformulation exhibited the ability to mitigate chronic inflammation, reduce oxidative stress, and promote tissue regeneration - all critical factors in diabetic wound healing. These effects can be attributed to the synergistic action of Rutin's intrinsic bioactivities and the enhanced delivery capabilities of the SLN platform. The observed improvements in wound closure rates, tissue remodeling, and angiogenesis underscore the therapeutic potential of RN-SLNs. By providing a localized, sustained release of Rutin, this nanoformulation overcomes many of the limitations associated with traditional topical treatments, such as rapid drug clearance and poor tissue penetration. Moreover, the favorable safety profile of RN-SLNs, as evidenced by histological and biochemical analyses, supports their potential for clinical translation. The absence of significant local or systemic toxicity is crucial for the development of long-term wound management strategies. In conclusion, Rutin-loaded Solid Lipid Nanoparticles represent a promising approach to diabetic wound management, offering a unique combination of targeted drug delivery and enhanced therapeutic efficacy. As research in this field progresses, such nanotechnology-based interventions hold the potential to significantly improve outcomes for patients suffering from chronic diabetic wounds, ultimately enhancing their quality of life and reducing the socioeconomic burden associated with this challenging condition.

6. DECLARATION OF INTEREST:

None.

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Received on 14.10.2024 Revised on 26.12.2024

Accepted on 13.02.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2826-2833.

DOI: 10.52711/0974-360X.2025.00405

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	Sample	Days
Group	Sample	1st	8th /0th of PW	8th-21st	21st / 14th of PW
Group-I	Vehicle (Control)	Administered Single I.P. Injection of STZ	Glucose Level Estimation and Wound Creation	-	Excised Wound Tissue
Group-II	Treated with Plermin Gel (Standard)	Administered Single I.P. Injection of STZ	Glucose Level Estimation and Wound Creation	Plermin Gel Given (Topically)	Excised Wound Tissue
Group-III	Treated with RN-gel	Administered Single I.P. Injection of STZ	Glucose Level Estimation and Wound Creation	RN-Gel Given (Topically)	Excised Wound Tissue
Group-IV	Treated with RN-SLNs-gel	Administered Single I.P. Injection of STZ	Glucose Level Estimation and Wound Creation	RN-SLNS-Gel Given (Topically)	Excised Wound Tissue

Group	% Wound contraction ; Post-wounding days				Epithelization period (Days)
Group	2^nd	4^th	8^th	14^th	Epithelization period (Days)
Group I	3.1±1.2	7.2±2.1	20±0.7	31±5.3	29.4±0.60
Group II	8.1±2.8	26.2±3.1	46.5±7.1	68.8±5.2	22.8±2.30
Group III	6.4±1.3	26.7±3.2	38.74±5.9	51.23±6.2	32.2±1.21
Group IV	9.3±3.3	31.5±2.5	48.5±7.7	78±4.1	18±0.3