Enhanced therapeutic approach for diabetic foot ulcers: comparative study and characterisation of mupirocin-loaded nanosponge gel with marketed formulation for accelerating wound healing and in vivo evaluation

Background The current investigation aimed to develop, optimise, and assess a mupirocin-loaded nanosponge-based topical delivery system for diabetic foot ulcer and to achieve prolonged drug release while improving drug deposition within the skin. The nanosponges carrying mupirocin were formulated using the emulsion solvent diffusion method. A 3 2 factorial design was utilised to investigate effect of two factors, specifically the concentration of ethyl cellulose and the stirring rate, on the physical attributes of the nanosponges. The optimised nanosponge formulation batch (F9) was subsequently incorporated into a Carbopol gel base, ensuring the desired physical attributes were achieved in the gel formulation containing nanosponges. The research included in vitro drug release evaluation, ex vivo drug deposition analysis, assessment of the antimicrobial action of the nanosponge formulation, and in vivo diabetic wound healing


Background
Diabetes mellitus (DM) encompasses a cluster of persistent metabolic disorders marked by high blood glucose levels stemming from either inadequate insulin production or resistance to its effects [1].The two clinically distinct types are type 1, caused by autoimmune beta-cell destruction resulting in complete insulin deficiency, and type 2, characterized by increased resistance to insulin action and insufficient insulin production [2].Type 2 Diabetes Mellitus (T2DM) is the predominant form of diabetes, accounting for over 90% of cases globally.It is characterised by low insulin production and tissue insulin resistance [3].The global prevalence of diabetes is rising, with projections estimating over 1.31 billion people will be affected by 2050 [4,5].
Diabetic foot ulcers (DFUs) are substantial complications of diabetes, distinguished by ulceration linked with neuropathy and/or peripheral arterial disease [6][7][8].DFUs can lead to severe complications such as infections, amputations, and mortality.Infections are observed in up to 58% of patients with new foot ulcers [9].The prevalence of DFUs is higher in males (4.5%) than in females (3.5%) and in type 2 diabetics (6.4%) compared to type 1 diabetics (5.5%) [10].Patients with diabetes experience varying foot sensitivity symptoms, ranging from pain and tingling in the early stages to numbness and toe weakening in the later stages [11].DFUs are challenging to heal due to the presence of microbial biofilms, elaborate societies of microscopic organisms encased in a self-generated matrix of extracellular polymeric substances (EPS) [12,13].Diabetic mouse models hold clinical significance in relation to diabetic ulcers, while the excision wound mouse model is pertinent to both acute and chronic wound healing.Interestingly, the excision wound healing model in mice has potential applicability in diabetic wound healing as well, achieved by inducing diabetes prior to initiating the wound [14].
Nanosponge-Based Topical Drug Delivery: Conventional drug therapy for DFUs faces limitations due to neuropathy, hindering drug delivery to the injured site.Nanotherapeutics, particularly nanosponge-based topical delivery systems, offer advantages for chronic wounds like diabetic wounds, promoting effective wound healing and skin regeneration [15].The proposed strategy involves preparing a mupirocin-loaded nanosponge topical dosage form capable of penetrating deeper tissue at the injured site.Mupirocin (Pseudomonic acid A), an antibiotic synthesised by Pseudomonas fluorescens, demonstrated pronounced efficacy against staphylococci and streptococci, as well as specific gram-negative bacteria like Haemophilus influenzae and Neisseria gonorrhoeae.Additionally, it supports wound healing by promoting keratinocyte proliferation and augmenting growth factor production.The therapeutic benefits of mupirocin (MP) can be enhanced by synergistically integrating it with other substances and implementing innovative approaches [16][17][18][19].Nanosponges (NSs) are nanosized sponge-like structures with numerous cavities capable of accommodating payloads [20].NSs exhibit self-sterilising properties due to their average pore size of 0.25 µm, effectively preventing bacterial penetration.Additionally, they enhance drug bioavailability and improve the solubility of poorly soluble drugs [21].They present several advantages over microsponges, including smaller particle size (below 500 nm), enhanced stability (up to 300 °C), and lipophilicity, which allows masking unpleasant flavours and transforming the physical state of drug from liquid to solid [22].This unique drug delivery technology holds promise for attaining controlled and extended drug release, addressing the challenges of conventional topical delivery systems for diabetic wound healing.

Fabrication of nanosponge (NSs)
The preparation of MP-loaded Nanosponge (MP-NSs) was done by the emulsion solvent diffusion (ESD) method.MP and EC were used in the ratios of 1:4, 1:6, and 1:8.The amount of MP was kept constant at 100 mg in the development of NSs.In the emulsion solvent diffusion method, the NSs were fabricated by incorporating different proportions of EC.The internal organic phase, consisting of MP and EC, was blended with 20 mL of dichloromethane (Alternative to DCM are Ethanol, Ethyl Acetate or Acetone) using ultrasonic agitation in an ultrasonicated bath (70 kHz frequency) for a duration of 2 minutes (Crest, Ultrasonic Corporation, Cortland, New York), and in the external aqueous phase of 100 mL of distilled water, 1 g of polyvinyl alcohol (PVA) was dissolved by heating up to 60 °C.Then the internal organic phase was dropwise added to the external aqueous phase while stirring at 1000 rpm for 2 hours (hrs), and the fabricated NSs were gathered by filtration followed by oven drying at 40 °C for 24 hrs [23].

Factorial experimentation and optimization using design of experiments (DoE) software
Initial experiments were conducted to investigate the effect of MP/EC ratios and stirring rates on the physical properties of NSs.Throughout all formulations, the concentration of MP, internal phase volume, and PVA concentration remained consistent.To optimise the dependent variables like production yield (PY), entrapment efficiency (EE), and mean particle size (MPS) of nanosponges, nine formulations were created using a 3 2 -factorial design, with the independent variables being polymer concentration (X1) and stirring rate (X2) [24].

Fourier transform infrared (FT-IR) spectroscopy analysis
FT-IR spectroscopy data of MP, EC, and optimised MP-NSs were recorded on an FT-IR spectrophotometer (Jasco FT-IR 6700) using the potassium bromide (KBr) press technique as per previously reported methods.Approximately 1-4 mg of sample was combined with dry KBr in a 1:1 ratio and scanned at transmission mode over 4000-400 cm −1 [25].

Thermal analysis by differential scanning calorimetry (DSC)
The thermal assessment of MP, EC, and MP-NSs was conducted using a differential scanning calorimeter (Mettler Toledo DSC, USA).Precisely measured quantities of samples (5 mg) were placed in aluminium containers and hermetically sealed.Each sample was subjected to a gradual temperature increase of 10 °C per minute within the temperature interval of 25-300 °C, all in a nitrogen environment [26].

Solid state characterisation
The X-ray diffraction (XRD) study was conducted to evaluate the solid-state character of the formulation.Powder XRD studies of MP, EC, and optimised MP-NSs were conducted using a powder X-ray diffractometer (Brucker D2 Phaser 2nd Gen).Samples were placed in the sample stage, and data were obtained over 2θ range from 5 to 50° using a step size of 0.019° per sec [27].

Particle size and zeta potential characterisation
The analysis of particle size for MP-NSs was executed employing the "Malvern Zetasizer NanoZS (Malvern Instruments, UK)".The specimen being investigated was diluted with distilled water (1:200) and introduced into a disposable polystyrene cuvette.The measurement of particle size and polydispersity index (PDI) was conducted based on the principles of dynamic light scattering (DLS).The identical procedure was adhered to for gauging zeta potential (ZP), albeit employing an electrode cuvette.Each sample was subjected to triplicate testing (n = 3) [28].

Entrapment efficiency (%EE)
The ultracentrifugation technique was used to assess the entrapment efficiency of MP-NSs.Samples were centrifuged at 10000 rpm for 30 min using an ultracentrifuge (Remi C-24, Mumbai, India).Unentrapped MP content in the supernatant was diluted with an appropriate medium before being measured using a UV-visible spectrophotometer at 222 nm [29].Entrapment efficiency was calculated as per Eq.(1).

Scanning electron microscopy (SEM) analysis
The structure of NSs was investigated utilising a scanning electron microscope (GEOL 5400, USA) with an operational voltage of 20 kV.Prior to observation, dehydrated NSs underwent a 45-s coating with a gold-palladium alloy in an argon atmosphere.The SEM image was captured at a magnification of 3000 [30].

Generation of bacteria inoculums
Inoculum was standardised, and 106 colony-forming units (CFU/mL) of the required density were achieved.Nutritious broth (5 mL) was mixed with a loopful of the Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the mixture was then cultured for 24 h at 3 °C.To standardise the culture to 106 CFU/ mL (equal to 0.5 McFarland standards), 0.2 mL of the microbes 24-h culture were poured into 20 mL of sterilised nutritious broth and cultured for 3-5 h.Cultures of test microbes on agar-agar and 8% nutritional broth were combined to create Nutritional Broth Medium (NBM), which was used to cultivate the bacterial strains.It was autoclaved at 15 lbs.pressure for 25-30 min.On petri plates, 15 mL of NBM were poured to prepare agar test plates in an aseptic condition, then subjected to stabilisation at room tempertature (RT).In peptone saline solution, bacterial cell cultures were routinely subcultured and incubated at 37 °C for 24 h.

Agar plates and test sample preparation
The bacterial strains were inoculated onto sterile agar plates by streaking the swab across the entire surface (1) weight of total drug − weight of free drug × 100 weight of total drug of the agar 2-3 times to ensure uniform distribution of the inoculum.The agar plate was rotated at a 60° angle during this process.Subsequently, the plates were allowed to air-dry in a sterile environment at RT. Wells with a diameter of 9 mm were then carefully created in the plates under aseptic conditions.The preparation of the test samples (MP-NSs) at a concentration of 10 mg/mL and the reference drug Ciprofloxacin at 100 µg/mL in dimethyl sulfoxide (DMSO) was carried out.Using a sterile micropipette, 50 μL of both the reference and test samples were dispensed into the wells.The plates were positioned in an incubator adjusted to a temperature of 37 °C for a duration of 24 h and the zone of inhibition (ZOI) for each bacterial strain was measured in triplicate using a calibrated digital Vernier caliper.

Agar well diffusion method
By using this method, the antibacterial action of MP-NSs formulation was tested against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.These three microorganisms are most abundant in DFUs [31].Sterile Muller Hinton plates of agar were formed, and wells of 6 mm were punched into the plates using a sterilised cup borer.The plates were swabbed with a 24 h culture of test organisms using sterile cotton swabs.To the wells, 100 µL of MP-NSs solution were added, and plates were subjected to incubation in an upward position at 37 °C for 24 h., after which the plates were checked for ZOI.Control was 100 µL of (10% v/v) DMSO solution.The experiment was performed in triplicate, and the mean of the zone sizes recorded was calculated.

Preparation of MP-loaded nanosponge gel
To prolong the retention time on the skin surface, an optimised formulation of MP-NSs was selected for conversion into a topical gel system.Carbopol-934 (0.5%w/v) was allowed to swell in double-distilled water for 12 h.The dry powder of MP-NSs (equivalent to 100 mg of the drug), propyl paraben (0.5%w/v), and methyl paraben (0.2%w/v) were added to 10 mL of propylene glycol, and the propylene glycol suspension was gradually added to the swelled CP-934 gel while continuously stirring to achieve a uniform mixture.To obtain a translucent gel, a 1:1 molar ratio of triethanolamine base and CP-934 was added to the homogenous mixture.This step was taken to ensure a well-mixed and visually appealing gel product [32].

Assessment of gel containing MP-nanosponges
Upon visual inspection of the gel for its texture, colour, and uniformity, a comprehensive evaluation was conducted on the following parameters:

pH measurement
The pH of the formulated gel was gauged using a pH metre that had been calibrated with a pH 7 buffer prior to use.The electrode tip was immersed in the gel, and the reading was taken after 2 min.This pH measurement procedure was performed in triplicate, and the average value was computed.

Spreadability analysis
The spreadability of the gel was determined by placing a known weight of the sample between two glass slides, and a weight of 500 g was applied over the slides for approximately 5 min, after which no further spreading was anticipated.The initial and final diameters of the spread circles were measured in centimetres, serving as comparative metrics for spreadability.

Viscosity measurement
Viscosity, which signifies a resistance of fluid to flow, was assessed using a Brookfield viscometer equipped with spindle No. 7, operating at various rotations per minute (rpm) at room temperature (RT).

Drug content analysis
1 g of MP-NSs gel was precisely weighed, dissolved in methanol, sonicated for 15 min, and then adjusted to the mark in a 100 mL volumetric flask using methanol.From this solution, 1 mL was withdrawn, further diluted to 10 mL with methanol, and then a final dilution was carried out using distilled water to achieve a concentration within the Beer's law range.The absorbance was measured at 222 nm using a UV spectrophotometer against a blank gel treated in the same manner as a sample [33].

In vitro drug release assay
In vitro drug release investigations were conducted using Franz diffusion cells with a receptor chamber capacity of 20 mL and an effective diffusion area of 3.14 cm2.A cellulose dialysis membrane from Himedia, Mumbai, India, was soaked in the receptor medium (phosphate buffer, pH 5.8) for a period of 24 h prior to the commencement of the experiment.The donor side of the arrangement held a predetermined quantity of gel containing MP-NSs.
During the experiment, the receptor solution was consistently agitated at 50 rpm and maintained at a steady temperature of 32 ± 0.5 °C through a circulating jacket.At specific intervals, 1 mL samples were withdrawn from the receiving compartment, and an equal volume of fresh buffer was introduced to ensure sink conditions.The extracted samples were subjected to analysis using a UV-Spectrophotometer to quantify the quantity of released MP.To facilitate comparison, the release profiles of a conventional cream (Mupirocin Cream USP, 2%, Glenmark) and a commercially available MP ointment (T-bact, GlaxoSmithKline) formulation were also examined.The drug release data underwent linear regression analysis to determine the release kinetics, encompassing zero-order and first-order release kinetics, as well as the diffusion-controlled mechanism (Higuchi model).[34].

Ex-vivo drug deposition assay
A study involving drug deposition within the skin was conducted using a Franz diffusion cell and excised rat abdominal skin.The outer layer of the skin was exposed to the surrounding environment, while the inner layer faced the solution in contact.The surface of the skin facing the outside environment was the epidermal side, whereas the dermal side was directed towards the solution in contact.The receptor compartment was filled with 20 mL of phosphate buffer at pH 5.8, maintained at a temperature of 33 ± 0.6 °C, and agitated at a rate of 50 rpm.Prior to the application of the sample, the skin was saturated with the diffusion medium for an hour.A 40-mg portion of the sample was applied to the donor compartment.For the quantification of the drug accumulated in the skin, the diffusion cell was disassembled after time intervals of 4, 8, 16, and 24 h.The skin was cautiously detached, and the mupirocin present on the surface was cleansed using distilled water [34].

Determining mupirocin concentrations in skin specimens
MP was recovered from the skin utilizing a technique outlined by Echevarria et al. [35].In brief, the skin was fragmented into smaller sections and subsequently crushed and ground using 10 mL of methanol.Following this, the crushed mixture underwent a 15-min session of ultrasonication to ensure the comprehensive extraction of the drug.The methanolic extract that resulted was later centrifuged at a speed of 8,000 rpm for a duration of 15 min.The liquid portion above the sediment, which held the extracted substances, was meticulously gathered.This collected supernatant was then evaporated and subsequently mixed back with the suitable solvent.Prior to analysis, the sample was filtered using 0.2-μm Whatman filter paper and subjected to assessment using a UV spectrophotometer at 222 nm.Intact skin was enriched with predetermined quantities of the drug to determine the recovery rate of the drug from the skin Subsequently, the skin was fragmented into smaller sections, crushed, drug extracted, and analysed using the previously described procedure.

Skin irritation studies
The optimised gel formulation containing MP-NSs was subjected to skin irritation assessments using the HET-CAM (Hen's Egg Test-Chorioallantoic Membrane) technique [36].Fertile white chicken eggs were sourced from commercial suppliers.Fresh eggs, which were nine days older and weighed between 50 and 60 g, were selected for the study.The irritation evaluation involved the negative control, which was sodium chloride (0.9%w/v NaCl), the positive control, which was sodium hydroxide (1%w/v NaOH), and the prepared MP-NSs gel.The HET-CAM test was used to assess irritation.The irritation scores from all treated groups were recorded at various time intervals, and the mean irritation score was calculated [37].

Stability studies
The MP-NSs gel was evaluated for its stability in an accelerated stability chamber (REMI) at three different temperatures (4 ± 2 ºC, 25 ± 2 ºC and 37 ± 2 °C) and 75% relative humidity (RH) for three months.The gel was evaluated for physical appearance, viscosity, pH, and Spreadability.Any change in appearance, pH, viscosity, or Spreadability of the stored MP-NSs gel was recorded [38].

In vivo wound healing activity
Wistar albino female rats in good health, weighing 180 and 250 g, were selected.The Institutional Animal Ethics Committee (Approval number IAEC/UDPS/2022/02/08) granted approval to the study protocol in accordance with Indian Committee for the Purpose of Control and Supervision of Experiments with Animals (CPCSEA) specifications.Throughout the study, animals were kept in a standard laboratory environment at a temperature of 25 ± 2 °C with a relative humidity of 44-56% and fed a standard diet and water.

Diabetes animal model
The described experimental protocol involved inducing diabetes mellitus in Wistar rats through the intraperitoneal (I.P.) delivery of streptozotocin (STZ) at a dose of 60 mg/kg body weight.Prior to the STZ injection, the rats were subjected to an overnight fasting period.
To safeguard pancreatic beta cells from excessive harm induced by STZ, niacinamide was administered intraperitoneally at a dosage of 120 mg/kg body weight, 15 min prior to the STZ injection.[39,40].The STZ was prepared in a 0.1 M citrate buffer with a pH 4.5 for the I.P. injection.After 72 h from the STZ injection, blood samples were collected from the rats using the retro-orbital method, which involves obtaining blood from the blood vessels located behind the eye socket, a commonly used technique in small laboratory animals like rats.To confirm the successful induction of diabetes mellitus in the rats, various parameters were measured.These included HbA1C (glycated haemoglobin), blood glucose levels, and CRP (C-reactive protein) levels.These measurements were taken both before the induction of diabetes (baseline) and after 72 h following the STZ injection.The changes in these parameters would indicate the development of diabetes mellitus in the rats [41][42][43].

Excision wound model
Every rat participating in the study was administered an intraperitoneal injection of thiopentone sodium at a dosage of 40 mg/kg to induce anaesthesia on the day designated for wound creation.The wound development procedure involved creating a rectangular pattern on the upper side of the rat's paw.Using a scalpel blade, a wound was created by separating a complete skin layer with a standardised dimension of 2 mm × 5 mm.The rats were then randomly distributed into various experimental groups for further study.The purpose of this experimental setup was likely to be to investigate wound healing or other related phenomena in response to different treatments or interventions [44].

Animal groups and treatment protocol
Rats were split into 6 groups (n = 5), with 5 rats in each group, and exposed to the subsequent treatment: Treatment was applied topically to treat excised wound area.This treatment was followed once a daily.

Determination of wound area, calculation of wound contraction
On predefined days 1, 4, 6, 8, 10, 12, and 16, a camera was used to document the progression of changes in the wound location.Image analysis software computed the wound area from the pictures.By using Eq. ( 2), the percentage (%) of wound closure can be calculated [45].
GraphPad Prism VIII was used to plot the graph showing the % of wound closure vs the number of days since the wound first developed.

Formulation and optimization of nanosponges
The process of formulating nanosponges is presently restricted in terms of its intricacy and expense.Although certain nanosponges available on the market are manufactured through the suspension polymerization method, an alternative technique, referred to as the ESD method, has exhibited potential for nanosponge production.The ESD method is characterised by its simplicity, reproducibility, and efficiency, rendering it a fitting approach for generating MP-NSs.A noteworthy advantage of this method is its ability to bypass the use of toxic solvents.In order to assess the impact of various variables on % entrapment efficiency, % production yield, and mean particle size of nanosponges, a factorial design was employed alongside analysis of variance (ANOVA).The factors under consideration included the concentration of EC and the stirring rate, which were investigated to uncover their effects on the aforementioned parameters.Table 1 presents the coded levels of concentration of EC and stirring rate that were utilised in the experimental design.These coded levels served as the basis for investigating the impact of independent variables on dependent variables.Table 2 provides an overview of how the independent variables influenced these specific response variables, shedding light on the relationships and effects within the experimental framework.The visual representation of the data is facilitated through the utilisation of two-and three-dimensional surface plots.These plots elucidate the relationships between variables and response outcomes, these insightful visualisations are presented in Figs. 1 and 2. The regression equation (Eq. 3) for %EE is as follows: where, A represents the EC concentration and B represents the stirring rate.The results revealed that the concentration of EC had a notable positive influence on the drug entrapment efficiency (%EE), meaning that increasing the EC concentration led to higher drug entrapment.On the other hand, the stirring rate had a significant negative effect on %EE, indicating that higher stirring rates resulted in lower drug entrapment.
(3) Y1 (%EE) = 83.50+ 5.28A − 0.7783B  For particle yield (PY), a linear regression equation (Eq.4) was generated, indicating that both the EC concentration and stirring rate had a positive influence: Regarding mean particle size (MPS), a polynomial regression equation (Eq.5) revealed that the EC concentration had a positive effect, while the stirring rate had a negative effect: Fit statistics for data analysis of response variables are given in Table 3.Based on the response surface methodology study, it was found that formulation (F9) performed better in terms of %EE, %PY, and MPS.Postanalysis confirmation at a two-tailed 95% confidence level is given in Table 4.As a result, the optimised nanosponges batch (F9) was selected for further characterisation studies and incorporated into a Carbopol gel base.The findings from the factorial design and regression equations provide valuable insights for optimising nanosponge formulations with enhanced properties for potential pharmaceutical applications.

Characterisation of nanosponges
FT-IR spectroscopy analysis was conducted on MP, EC, and the optimised MP-NSs formulation (F9).The resulting data, as depicted in Fig. 3, demonstrated that the key peaks of MP were similarly present in the physical combination of MP, EC, and CP.This outcome suggests the absence of substantial alterations or interactions among these components within the mixture.Consequently, this observation indicates the stability of the formulations.
Additionally, the FT-IR spectrum (as illustrated in Fig. 4) of the optimised nanosponge formulation (F9) exhibited all the significant peaks corresponding to EC, whereas the major peaks attributed to MP were conspicuously absent.This observation serves as confirmation that the MP has been effectively encapsulated within the nanosponges.
In order to investigate thermal characteristics, thermograms were acquired for MP, EC, and the optimised MP-NSs formulation, as depicted in Fig. 5. MP exhibited a distinct endothermic peak at 77.78 °C, signifying its melting point.Notably, EC and the optimised MP-NSs formulation displayed a broad exothermic peak at the same temperature, which indicated the absence of the original The XRD graph (shown in Fig. 6) for MP displayed distinct sharp peaks, indicative of its crystalline structure.Conversely, both EC and the optimised MP-NSs    exhibited an amorphous nature in the XRD pattern, providing additional verification of the successful encapsulation of the MP within the nanosponges.Moreover, the optimised MP-NSs were characterised by a mean particle size of 189 nm and a zeta potential value of (− ) 26 mV, as depicted in Fig. 7. SEM images of the MP-NSs, as shown in Fig. 8, revealed a consistent spherical morphology characterized by a porous structure.Notably, these images displayed no discernible intact Mupirocin crystals, providing strong visual evidence of the effective entrapment of the drug within the nanosponge matrix.
In an antimicrobial study, the antimicrobial efficacy of the MP-NSs formulation was assessed against commonly found bacterial strains (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) associated with diabetic foot ulcers.Employing the agar-well diffusion method, inhibitory potential of nanosponge formulation was determined.Figure 9 illustrates these results, which were then compared with those of the reference drug Ciprofloxacin and a control solution.Recorded zone of inhibition values against the targeted microorganisms are compiled in Table 5.To visually represent the antimicrobial activity of MP-NSs, Fig. 10 offers a graphical depiction.This dataset provides valuable insights into the extent of inhibition exerted by the tested substances on the growth and activity of the selected microorganisms.
Taken together, the results from FT-IR, thermal analysis, SEM, and XRD studies strongly support the successful preparation of MP-NSs, wherein the drug is effectively entrapped in the nanosponge matrix, converting it from a crystalline state to an amorphous state.This formulation demonstrates promising potential for pharmaceutical applications due to its stability and drug entrapment capabilities.

Characterisation of nanosponges (MP-NSs) loaded gel
The developed carbopol gel formulations exhibited a uniform and smooth white texture, showcasing thixotropic properties that facilitated easy and efficient spreading.The pH value of the gel was quantified as 6.4 ± 0.03, and its viscosity was determined to be 1026 centipoises (cPs).Notably, the spreadability assessment of the gel containing MP-NSs yielded a value of 31.0 g•cm/sec, affirming its effective spreading capability.The drug content analysis revealed a value of 88.48 ± 0.04, indicating a uniform distribution of the drug within the gel matrix.These comprehensive findings are detailed in Table 6.

Skin irritation studies
The irritation assessment of the control with (0.9%w/v) NaCl, the control containing (1% w/v) NaOH, and the formulated MP-NSs gel was conducted through the utilisation of the HET-CAM test.Figure 11 illustrates the effects on blood vessels caused by different substances applied to the chorioallantoic membrane within a 5-min interval.The evaluation of the scores from all the treated groups was performed at various time intervals, and the average irritation scores are presented in Table 7.The negative control sample exhibited a score of zero at each time interval (signifying absence of irritation, ranging from 0 to 0.8), while the positive control demonstrated an exceedingly high score (16.43).Similarly, the formulated MP-NSs gel also resulted in a score of zero, indicating an absence of irritation on the chorioallantoic membrane.The absence of irritation scores in the negative control and the formulated solution suggests that they do not cause irritation to the membrane, thus establishing their non-irritating nature to the skin.

In vitro drug release assay
The impact of composition and carrier on the drug release pattern of distinct formulations were examined using a cellulose dialysis membrane.The in vitro discharge patterns of MP from diverse formulations, as depicted in Fig. 12, demonstrated that MP ointment and MP cream released the MP within 4 and 10 h, respectively.In contrast, the MP-NSs gel exhibited a prolonged release extending up to a 24-h period.The release of the MP from the gel formulation was governed by a diffusion-controlled mechanism.The percentage of drug release is outlined in Table 8.

Ex vivo drug deposition assay
The quantity of mupirocin accumulated in excised rat abdominal skin through various formulations at distinct time intervals is illustrated in Fig. 13.The quantity of mupirocin deposited within the skin was notably greater when using the MP-NSs gel (211.4 ± 6.9 μg/cm 2 ) in comparison to the MP cream (83.57± 6.7 μg/cm 2 ) or MP ointment (34.03 ± 5.6 μg/cm2) after 24 h.This observation underscores that the nanosponges facilitated an enhancement in the drug residence in the skin.

Stability study
The results of the stability study, as shown in Table 9, revealed that the MP-NSs gel was found to be stable after a period of three months.Significant changes have not been seen when these were demonstrated for physical appearance, pH, and drug content at different temperature conditions (4 ± 2 ºC, 25 ± 2 ºC and 37 ± 2 ºC).A drug

In vivo wound healing study of MP-NSs gel and marketed formulation
DM was induced in rats through the I.P. delivery of STZ at a dosage of 60 mg/kg body weight.Blood samples were obtained using the retro-orbital method both prior to and subsequent to the STZ injection.These samples were subsequently analysed for blood glucose levels, C-reactive protein (CRP) levels, and glycated haemoglobin (HbA1C) levels, as documented in Table 10.The outcomes of these assessments provided confirmation of the successful induction of diabetes.Meanwhile, an excision wound model was employed to create wounds on the rat feet.These wounds were then exposed to various distinct treatments for further investigation and analysis.Table 11 presents the results of the wound closure percentages for different treatment groups.The diabetic wounds treated with standard Becaplermin gel exhibited the highest percentage of wound closure (96%), outperforming all the other groups.The diabetic wounds treated with MP-NSs gel showed a commendable wound closure rate of 92%, which was higher than the diabetic wound control group (43%), the normal wound control group (73.5%),DM group 4 treated with MP ointment (79%), and DM group 5 treated with MP cream (78%).The bar graph in Fig. 14 illustrates the percentage of wound closure on days 4, 8, 12, and 16 for all the groups.On day 4, there was a consistent and exponential wound healing rate observed across all groups.However, from day 8 onwards, both DM group 3 (diabetic wound treated with MP-NSs gel) and DM group 6 (diabetic wound treated with Becaplermin gel following MP-NSs gel) exhibited enhanced wound healing compared to the rest of the groups.Figure 15 clearly demonstrated that the diabetic wounds treated with Becaplermin gel exhibited a faster rate of healing compared to all the other groups.The diabetic control group, which did not receive any treatment, showed the slowest healing rate, even when compared to the normal wound control group.Surprisingly, the group with diabetic wounds treated with MP-NSs gel showed earlier and more improved wound healing compared to both the diabetic control group and the normal wound control group.Additionally, when compared to standard MP ointment and MP cream, the MP-NSs gel displayed faster wound closure.This difference in efficacy can be attributed to the sustained release property of the nanosponge-based gel.While the marketed conventional products required thrice-daily application, the MP-NSs gel was administered only once a day due to its sustained release characteristics, leading to superior results in wound healing.Overall, these findings strongly suggest that the MP-NSs gel had a significantly positive effect on accelerating wound healing in diabetic rats.Its sustained release properties not only contributed to better efficacy compared to the conventional products but also facilitated once-daily delivery, simplifying the treatment protocol.These findings demonstrate that the MP-NSs gel was highly effective in promoting wound closure in diabetic rats, with a significant improvement over the untreated diabetic wounds and the control groups treated with MP ointment and MP cream.The standard Becaplermin gel displayed the highest wound closure percentage, but the MP-NSs gel showed a promising and competitive efficacy, making it a potential alternative treatment option for diabetic wound healing.

Discussion
The study aimed to develop Mupirocin-loaded nanosponges using the emulsion solvent diffusion method, an alternative and promising approach for nanosponge preparation.This method offers advantages such as simplicity, reproducibility, and rapidity, while also  Regarding MP entrapment efficiency (%EE), increasing the EC concentration positively impacted %EE, meaning that higher EC concentrations led to improved drug entrapment within the nanosponges.Conversely, higher stirring rates had a negative effect on %EE, indicating that excessive stirring negatively influenced drug entrapment.For production yield, both EC concentration and stirring rate had a positive influence, contributing to higher yields of nanosponges.The MPS of the nanosponges was influenced positively by EC concentration but negatively by stirring rate.This indicates that higher EC concentrations and lower stirring rates resulted in larger nanosponge particle sizes.
The FT-IR analysis provided insights into the interactions between mupirocin, ethyl cellulose, and carbopol, confirming the stability of the formulations.The thermal analysis and X-ray diffraction (XRD) data confirmed the successful entrapment of mupirocin within the nanosponges, converting it from a crystalline to an amorphous state.The nanosponge formulation (F9) showed promising properties, including an MPS of 189 nm and a zeta potential of (− ) 26 mV, indicating a stable and effective nanosponge formulation.The nanosponge-gel exhibited sustained drug release for up to 24 h, providing potential advantages in controlled drug delivery.The nanosponge formulation demonstrated improved drug deposition in excised rat abdominal skin compared to conventional formulations like ointment and cream.The HET-CAM irritation study indicated that the nanosponge-gel was non-irritant, making it a safe formulation for topical application.The stability study revealed that the nanosponge-gel remained stable over a three-month period, with a drug entrapment efficiency greater than 90%, suggesting the potential for long-term shelf life.
The in vivo study on diabetic rat wounds demonstrated that the nanosponge-gel significantly accelerated wound healing compared to conventional products and even outperformed MP ointment and MP cream.The sustained release property of the nanosponge-gel facilitated once-daily delivery and provided improved wound closure efficacy.while the gel fostered an optimally humid environment conducive to wound care during the final stages of wound healing and sealing [46].
The acceleration of wound healing is primarily attributed to Mupirocin, which plays a pivotal role in stimulating the production of growth factors and the proliferation of human keratinocytes [18].Additionally, the nanosponges, due to their smaller size, exhibit enhanced penetration capabilities into deeper tissues at the injured site.The gel component of the formulation fosters an optimally humid environment, particularly beneficial during the final stages of wound healing and sealing.Therefore, the combined efficacy of Mupirocin-loaded nanosponge  gel as a dosage form significantly contributes to the overall acceleration of the wound healing process.Overall, the results of this study indicate successful development of mupirocin-loaded nanosponges using the emulsion solvent diffusion method, providing a potential alternative for wound healing in diabetic rats.The nanosponge-gel exhibited favourable properties, including sustained drug release, enhanced drug deposition within the skin, stability, and non-irritating characteristics.This novel formulation has promising potential for pharmaceutical applications and merits further investigation for potential clinical translation as an effective and convenient treatment option for wound healing.

Conclusion
A novel drug delivery system utilising mupirocin-loaded nanosponges has been successfully developed to facilitate once-a-day sustained release medication for the topical treatment of diabetic wounds.The innovative formulations demonstrated improved drug retention within the skin, showcasing the superior potential of the nanosponge-based delivery system when compared to conventional mupirocin ointments and creams available in the market.Considering the heightened effectiveness and the enhanced patient adherence due to reduced application frequency, it is evident that the nanosponge-based gel formulations will play a substantially more beneficial role in the treatment of diabetic wounds.

Group I :
Control group with normal wound (NWC), Non-Diabetic animal with wound received citrate buffer and distilled water.Group II: Control group with Diabetic wound; Diabetic animals with wounds who received no treatment (DWC).Group III: Diabetic wound treatment by MP-NSs gel formulation.Group IV: Diabetic wound treatment by standard MP ointment (T-bact, GlaxoSmithKline).Group V: Diabetic wound treatment by standard MP Cream (Mupirocin Cream USP, 2%, Glenmark).Group VI: Diabetic wound treatment by standard Becaplermin gel (REGRANEX, Smith&Nephew).

( 2 )
% Wound closure = [Area of initial wound) − N th day area of wound × 100 (Area of initial wound)

Fig. 1
Fig. 1 Three-dimensional surface plots of A Entrapment efficiency, B Particle size, C Production yield and D Desirability plot

Fig. 7 Fig. 8
Fig. 7 Particle size A and zeta potential B of MP-NSs

Fig. 12
Fig.12 In-vitro drug release of marketed formulation and prepared mupirocin-loaded nanosponge-gel formulation

Fig. 13
Fig.13 Graphical representation of concentration of drug deposited in rat skin at different time intervals

Table 1
Coded level of ethyl cellulose and stirring rate for experimental design

Table 2
The effect of Mupirocin: Ethyl cellulose ratio and stirring rate on production yield, EE, and mean particle size α Each observation is the mean ± SD of three determinations

Table 3
Fit statistics for data analysis of response variables using full factorial design

Table 4
Post Analysis confirmation at two tailed, 95% confidence level

Table 8
Percent (%) drug released of marketed mupirocin ointment, mupirocin cream and prepared mupirocin-loaded nanosponge gel formulation α Each observation is the mean ± SD of three determinations

Table 9
Results of stability studies of MP-NSs gel

Table 10
Confirmation of induction of diabetes mellitus in rats

Table 11
Tabular representation of percentage wound closure whereas NWC Normal wound control, DWC Diabetic wound control, DW Group 1 Diabetic wound treated with mupirocin-loaded nanosponge gel, DW Group 2 Diabetic wound treated with MP-ointment, DW Group 3 Diabetic wound treated with MP-cream, DW Group 4 Diabetic wound treated with Becaplermin gel