A stimuli-responsive in situ spray hydrogel co-loaded with naringenin and gentamicin for chronic wounds

Thaneswary Thangarajoo; Yong Kai Hsin; Manisha Pandey; Hira Choudhury; Lim Wei Meng; Shadab Md; Md Habban Akhter; Bapi Gorain

doi:10.1515/chem-2022-0357

Article Open Access

A stimuli-responsive in situ spray hydrogel co-loaded with naringenin and gentamicin for chronic wounds

Thaneswary Thangarajoo , Yong Kai Hsin , Manisha Pandey , Hira Choudhury , Lim Wei Meng , Shadab Md , Md Habban Akhter and Bapi Gorain

Published/Copyright: July 20, 2023

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From the journal Open Chemistry Volume 21 Issue 1

Abstract

The potentials held by stimuli-responsive polymers in wound dressing have led to the present research in formulating a hydrogel base formulation with polymers having pH and thermo-sensitivity. Thus, hyaluronic acid (pH-sensitive polymer), and Pluronic F-127 (thermo-sensitive polymer) with hydroxypropyl methylcellulose (mucoadhesive polymer) were incorporated to obtain an in situ hydrogel containing gentamicin and naringenin (NAR). The optimization of the stimuli-responsive formulation was performed by the Box–Behnken statistical design to acquire variable parameters that influence the gelling temperature and viscosity. Thermo-gravimetric analysis, differential scanning calorimetry, and Fourier-transform infrared spectroscopy were performed to confirm the suitability of incorporating the selected polymers with drugs. The optimized formulations (blank and drug-loaded) were found to possess satisfactory characteristics of gelling temperatures (30–33°C), viscosities (174 ± 3 to 184 ± 4 cP), and mucoadhesive properties (0.29 ± 0.01 to 0.31 ± 0.01 N) with a spray diameter of 16.8 ± 1.4 to 18.9 ± 1.2 cm² to facilitate the application at the wound environment. The in vitro drug release study depicted a sustained release profile over a time frame of 8 h with a cumulative release of 56.18 ± 4.59% NAR. The drug-containing in situ hydrogels showed superior potency by producing a larger zone of inhibition (2.03 ± 0.12 cm). Furthermore, a cytotoxicity study of the developed formulations in HaCaT cells revealed no toxicity of the drug-loaded formulations when compared to the blank hydrogel. These findings indicate the potential of the in situ hydrogel as an effective wound dressing for chronic wounds; however, additional investigation is needed for further implementation.

Keywords: stimuli-responsive hydrogel; gentamicin and naringenin; Box–Behnken statistical design; zone of inhibition; cytotoxicity; application to chronic wound

1 Introduction

The normal healing process of wounds involves four overlapping stages – hemostasis, inflammation, tissue regeneration, and remodeling phase. However, during chronic wounds, the body’s healing process is disrupted [1], and instead of following these usual steps, the healing process discontinues at the inflammatory phase. In developed countries, approximately 1–2% of people suffer from chronic wounds, including diabetic foot ulcers, venous ulcers, and pressure ulcers [2]. In the United States, approximately 6.5 million of the population experience chronic wounds every year [3]. The economic burden to society on the management of chronic wound conditions is increasing proportionately to the increasing number of patients [1]. Improper management of the chronic wound can worsen to end up undergoing amputation of their extremities, which affects the quality of life of the patient and can lead to death too. However, conditions of a chronic wound can be coped with by focusing, managing, and treating the indicators that determine the wound severity, which include elevation of pH and temperature, reduction of moisture level and oxygen supply, etc., which facilitate bacterial growth [4]. Continuous research in this field is in progress to develop proper and effective wound dressings in a cost-effective manner, which could help in promoting complete healing [5,6,7,8,9]. Therefore, the development of an ideal wound dressing is an urgent need that will not only heal the wound but also possess the ability to retain at the site of action with controlled release of therapeutics, keep moistened wound environment, allow gaseous exchange, and prevent the entry of pathogens or external contaminants [2]. Among the explored platforms, hydrogel has been established as one of the potential dressings, which could possess all the essential properties of an ideal dressing such as adherence, prolonged delivery of drugs, enhance wound healing, etc.; therefore, it has been classified as one of the promising tools in wound dressings. The entrapped drugs are released at a controlled rate for a longer period to provide the essential components continuously to heal the wound in a better way [10]. Advancement of research in this field demonstrated the enhancement of self-healing properties [8,11], whereas a few studies have been carried out with the incorporation of engineered materials into the hydrogel dressings for improved properties [5,12,13,14,15].

Recent research with novel functional polymers using the facilities of fabricating a stimuli-responsive tool due to their functionality and responsivity towards a particular environment is the most surge field. Structural deformation of the polymers due to the exposure of stimuli at the application site allows the development of a “smart” device, which actuates when applied to the wound environment [16]. The role of stimuli-responsive polymers has been explored widely for three types of polymers, chemical, physical, and biochemical. These polymers are further divided into specific sensitivities such as pH-sensitive, thermo-sensitive, ion-sensitive polymers, and many more [16]. Alteration of the properties of these polymers due to the stimuli at the applied site modifies their swelling property, viscosity, mucoadhesiveness, and porosity, which could change the state of the hydrogel, enhancing drug delivery [17,18]. The combination of two or more stimuli-responsive polymers in a single platform is a novel approach to advance actuation for better outcomes. For example, Wu et al. incorporated quaternized chitosan with glycerophosphate in which they fabricated thermo- and pH-sensitive polymers in delivering ocular-injectable formulation and it was shown to respond to temperature by swelling at 37°C and at neutral to basic conditions, in which the drug was released with good control [19]. The advancement of stimuli-responsive actuators is also explored in enhancing the drug delivery system for treating chronic wounds, where the modified environment alters the release characteristics of the incorporated therapeutic agents in the dressings to facilitate prevention and eradication of the infection faster [20].

Therefore, with the aim of developing an effective dressing for chronic wounds, the present research focuses on formulating a hydrogel formulation of two therapeutics – gentamicin and naringenin (NAR) – with two stimuli-responsive polymers – pH-responsive polymers, and thermo-sensitive polymers. Hyaluronic acid, a polyacid pH-sensitive polymer, swells in the basic environment [21]. Therefore, it would be suitable to be incorporated into the alkaline environment of the chronic wound [7]. Alternatively, Pluronic F-127 was selected as a thermo-sensitive polymer, which transits from sol to gel at the wound environment at a particular concentration. Additionally, the incorporation of a mucoadhesive polymer could enhance the adhesive property of the formulation to facilitate longer retention at the site of application. Furthermore, the presence of suitable antimicrobial agents in dressings enhances the healing process. Gentamicin is one of the antimicrobial agents that is commonly used in the treatment of wounds. It is classified as an aminoglycoside and it is a broad-spectrum antibiotic that covers a wide range of bacteria [22]. Topical administration of gentamicin is advantageous in the fact that it would not damage kidney function, lowers the risk of resistance development towards pathogens, and has a good ability in killing invading bacteria. Research had revealed that gentamicin-impregnated collagen sponge on the diabetic wound could shorten the duration of healing in diabetic patients, where the authors reported that the healing could be achieved within 2 weeks of application [23]. Additionally, recent research on wound healing is also focusing on the use of natural components that possesses beneficial efficacy, which could help in preventing infection and advance the progress of wound healing. NAR (5,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one), a flavanone, found primarily in citrus fruits and tomatoes, is known to possess lots of beneficial properties for our health issues, such as anti-inflammatory, antioxidant, and antimicrobial activities [5]. Such activities are essential for a good candidate in wound healing, as depicted in recent research that there was an increase in wound contraction, an effective healing index with shortened epithelialization period by the use of NAR formulation [5,24]. Therefore, the development of an optimized smart hydrogel system using thermo-reversible and pH-responsive polymers was achieved using Box–Behnken statistical design, where three different formulations were fabricated with gentamicin sulfate and NAR to comparatively study their performances. Furthermore, studies were performed to determine the lack of interaction between the polymers and drugs, and optimized formulations were characterized for their suitability for application in the wound environment. In addition, a microbial test was conducted using the disc diffusion method to establish the superiority of our developed formulation compared to the commercial one in wound healing. Finally, the cytotoxicity of the formulation was determined using human keratinocyte (HaCaT) cells to further prove the applicability of the formulation in the wound environment.

2 Materials and methods

2.1 Materials

NAR with a purity of >95%, hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC), Carbopol 934, Carbopol 940, Pluronic F-127, Pluronic F-68, and hyaluronic acid sodium salt were obtained from Sigma Aldrich (St. Louis, MO, USA). Gentamicin sulfate was gifted by Y.S.P. Industries (M) Sdn Bhd (Bangi, Selangor Darul Ehsan, Malaysia). Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and antibiotics (streptomycin and penicillin) were procured from Life Technologies (USA). The HaCaT cells were obtained from CLS-Cell Line Services (Germany). Purified distilled water was used in the experiment.

2.2 Methodology

2.2.1 Selection of suitable polymers for the preparation of in situ hydrogel preparation

2.2.1.1 Selection of a suitable mucoadhesive polymer

Based on the literature, dispersions of selected concentrations of HPMC (0.1–0.6% w/v), CMC (0.1–0.4% w/v), Carbopol 934 (0.1–0.6% w/v), and Carbopol 940 (0.1–0.6% w/v) were prepared by dispersing the polymers in 10 mL of distilled water. The dispersions were made by vigorous stirring using a vortex mixer for 15–30 min until the polymers were completely dispersed. The tubes were then kept overnight at room temperature and the viscosity and texture were evaluated by immersing a glass rod in each tube and examining the tip of the glass rod.

2.2.1.2 Selection of a suitable thermo-responsive polymer and pH-responsive polymer

Pluronic F-127 and Pluronic F-68 are usually incorporated in the formulation as thermo-sensitive polymers, which have the property to change into a gel upon the change in temperature. A few selected concentrations between 15 and 20% (w/v) for Pluronic F-127 and 9 and 13% (w/v) for Pluronic F-68 were dispersed into cold distilled water (4–5°C) and mixed vigorously using a vortex mixer for 15–30 min until the polymers were completely dispersed. Thereafter, the tubes were kept overnight in the refrigerator maintained at 2–4°C. The particular polymer and the respective concentration were chosen based on the determination of the temperature of the gelling point of the polymer dispersions (Section 2.2.2). On the other hand, concentrations of hyaluronic acid for the hydrogel system were selected between 0 and 0.5% based on the literature and the observed findings of the polymer dispersions.

2.2.2 Determining the temperature gelling point

The test tube inversion method was used to carry out the temperature gelling test [25]. The temperature gelling point method was done by immersing a glass test tube (diameter, 10 mm) in 1 mL of the formulation containing polymers at the body temperature. A thermometer was used to check the gradual rise of temperature in the test tube. The change in consistency was checked with the rise of every degree temperature starting from 25°C. When the solution appeared to be a gel-like consistency, the particular temperature was recorded and the same experiment was repeated thrice for a single concentration.

2.2.3 Optimizing a suitable concentration of the combination of pH-responsive, thermo-responsive, and mucoadhesive polymers

The Box–Behnken statistical design (Design Expert^®, version 12; State-Ease Inc., USA) was used with three factors at their three levels to optimize the stimuli-responsive blank hydrogel formulation, where the concentrations of pH-responsive (hyaluronic acid), thermo-responsive (Pluronic^® F127), and mucoadhesive (HPMC) polymers were considered as independent variables. Based on the reported data and the obtained results of the preliminary experiments, the highest and lowest concentrations of three different polymers were selected as high and low levels of the independent variables. Upon inclusion of low (−1), medium (0), and high (+1) levels of three different polymers, the Design of Expert experimental model suggested 15 different formulations with different combinations of three polymers at their different levels, which are presented in Table 1. Thereafter, the suggested formulations were developed and optimized by using three independent and two dependent variables (gelling temperature and viscosity). The design of experiment software was used for optimization, and statistical analysis was performed using analysis of variance (ANOVA). Finally, the effect of independent variables on the dependent variables was assessed using the polynomial quadratic equation (1) and the generated 3D surface plots, contour plots, and perturbation plots [5,26]. The software suggested polynomial quadratic equation (1) is presented henceforth:

(1) Y = b 0 + b 1 A + b 2B + b 3C + b 12AB + b 13AC + b 23BC + b 11A2 + b 22B2 + b 33C2 ,

where the measured response is represented by Y, b ₀ is the intercept, and b ₀ to b ₁ represent the regression coefficients for the model terms of A, B, and C, respectively.

Table 1

Independent and dependent variables used in the Box–Behnken design along with the generated experimental runs and corresponding experimental values

Run	Independent variables			Dependent variable
Run	X1: HPMC (% w/v)	X2: Pluronic F-127 (% w/v)	X3: Hyaluronic acid (% w/v)	Viscosity (cP)	Gelling temperature (°C)
1	−1	0	−1	120	35.5
2	−1	0	1	340	34.5
3	0	−1	−1	160	38.0
4	1	−1	0	280	36.0
5	0	1	−1	240	33.0
6	1	0	1	380	33.5
7	1	0	−1	280	35.0
8	1	1	0	340	30.5
9	0	0	0	240	34.5
10	0	1	1	420	31.5
11	−1	−1	0	180	37.2
12	0	0	0	220	34.8
13	0	−1	1	380	36.5
14	−1	1	0	260	32.5
15	0	0	0	240	34.0

Independent variable	Low (−1)	Medium (0)	High (+1)
A: HPMC	0.4%	0.5%	0.6%
B: Pluronic F-127	18%	18.5%	19.0%
C: Hyaluronic acid	0%	0.05%	0.1%

Dependent variable
Viscosity
Gelling temperature

2.2.4 Incorporation of gentamicin and NAR into the optimized in situ hydrogel formulation

Before the incorporation of the drugs into the formulation, the blank in situ hydrogel was prepared using the optimized ratio of three polymers as obtained from the software. Therefore, 0.5122% HPMC, 18.824% Pluronic F-127, and 0.07625% hyaluronic acid were dispersed in an aqueous environment and kept overnight at 2–4°C. Thereafter, the drug solutions were incorporated into this blank hydrogel.

Gentamicin sulfate is soluble in water; hence, the desired quantity of gentamicin was mixed directly into the blank formulation with vigorous stirring to obtain 0.3% w/v. On the other hand, NAR is insoluble in water but soluble in organic solvents. A 5% NAR solution was prepared by dissolving NAR in a solution containing 1:1 ethanol and dimethyl sulfoxide using a vortex mixer. Thereafter, the calculated quantity of the solution was transferred to the mixture of the three polymers and gentamicin to finally obtain a 0.5% NAR concentration. Thereby, four different formulations were fabricated: blank (without drugs) hydrogel, NAR-loaded hydrogel, gentamicin-loaded hydrogel, and a combination of hydrogel containing NAR and gentamicin to compare their performances at their characterization parameters.

2.2.5 Evaluation of interaction studies between polymers and therapeutic agents

2.2.5.1 Thermogravimetric analysis (TGA)

TGA was performed using a TGA 8000 PerkinElmer. Samples, gentamicin, NAR, hyaluronic acid, Pluronic F-127, and HPMC were heated in an aluminum pan at 20°C/min under a constant nitrogen flow rate of 20 mL/min. The NAR and hyaluronic acid were heated from 30 to 800°C [27], gentamicin sulfate was heated from 30 to 600°C [28], and HPMC and Pluronic F-127 were heated from 30 to 500°C.

2.2.5.2 Differential scanning calorimetry (DSC) analysis

All five samples, gentamicin, NAR, hyaluronic acid, Pluronic F-127, and HPMC were analyzed using a DSC instrument (DSC 1 Star System, Mettler Toledo, Bristol, UK). Based on the TGA of all individual samples, the DSC analysis was set to run between 25 and 400°C at a heating speed of 10°C/min under a nitrogen flow rate of 20 mL/min. A mixture of all samples in the ratio 1:1:1:1:1 was prepared and analyzed at a similar range of temperature as other samples, with similar speed under a similar nitrogen flow rate.

2.2.5.3 Fourier-transformed infrared (FTIR) spectroscopic analysis

An FTIR spectrophotometer (Spectrum 100; Perkin Elmer, Beaconsfield, UK) was used to analyze and obtained the FTIR transmission spectra of individual samples and a mixture thereof at a ratio of 1:1:1:1:1 in a dry form. FTIR analysis of gentamicin sulfate, hyaluronic acid, and NAR was carried out by preparing potassium bromide (KBr) pellets at a ratio of 2:98, where a thin pellet was formed by compressing with a hydraulic pump. The pellet was then placed in the IR beam of the spectrophotometer and it was scanned within a range of 4,000–450 cm⁻¹ [29,30]. The spectra of Pluronic F-127, HPMC, and the mixture of all five samples were analyzed in the attenuated total reflectance of the FTIR spectrophotometer in the range of 4,000–450 cm⁻¹ at a resolution of 4 cm⁻¹ [31].

2.2.6 Characterization of the drug-loaded in situ hydrogel preparation

2.2.6.1 Determination of pH and the viscosity test

The pH of the blank and different drug-loaded formulations was measured by using a Sartorius PB-10 pH meter at 25°C. The viscosity of all the prepared formulations was measured using a Brookfield viscometer (DV2T) with spindle size 63 (LV-03) at 34°C, i.e., the temperature of the skin environment. All the experiments were performed in triplicate [7].

2.2.6.2 Spray dimension test

As outlined by the United States Food and Drug Administration (FDA), the characterization of the spray pattern is commonly performed as a quality control test to qualitatively evaluate the performance of actuators and respiratory formulation development [32]. The four optimized formulations were evaluated for their spray dimension to determine the coverage area of the formulation with one spray. The formulations were transferred into empty spray bottles. The spray dimension test was performed based on the research conducted by Gholizadeh et al. with a slight modification, where the spray coverage was determined by spraying the formulation on a graph paper from a 7 cm distance on a vertical platform maintained at 34°C [33]. The formulation was manually sprayed by a single actuation, and the dimension of the area covered with the formulation was calculated mathematically after drying the graph paper.

2.2.6.3 Mucoadhesive test

The mucoadhesive property was determined using the modified balance method [5]. The freshly excised goatskin was collected from the slaughterhouse and the hairs were shaved carefully; the skins were soaked in phosphate buffer saline (pH 7.4) and it was attached to the base of the weighing balance at one end. One milliliter of the respectively prepared formulations was placed on one side, which was attached to the base part “B” as shown in Figure 1. The temperature of part “B” was maintained at 37°C with the help of warm water. The side with the attached skin was brought into contact with the surface of the parafilm, where the formulation has been placed (E). On the other side of the balance, the weight was increased gradually. The mucoadhesive strength of a particular formulation was determined by the weight required to detach part “D” from part “B.”

Figure 1

Modified balance for the mucoadhesive test. (A) balance, (B) beaker with parafilm on the surface of the bottom of the beaker, (C) double-sided tape, (D) animal skin, (E) sample, and (F) weight.

2.2.6.4 In vitro drug release test using the Franz diffusion cell method

The Franz diffusion cell method [34] was used to carry out the in vitro drug release study of the prepared formulations, NAR and formulation loaded with gentamicin and NAR. Phosphate buffer (pH 7.4) was used as release media in the present experiment, which was transferred into the receptor chambers of the Franz diffusion cell. A piece of cellulose acetate membrane was placed at the opening of the receptor chamber, followed by the fixing of the donor chamber [35]. About 1 mL of each fabricated formulation was transferred into each donor chamber and the opening of the donor chamber was covered with parafilm to avoid contamination. Magnetic beads were placed in the receptor chamber to ensure continuous stirring of the solution. The temperature of the cell was maintained at 33°C, which is the mean temperature of the wound bed, by circulating the water jacket. A volume of 0.5 mL was withdrawn from the chambers via their sampling arm at particular time intervals and was then replaced with 0.5 mL of fresh phosphate buffer maintained at the same temperature after every withdrawal. Samples were withdrawn at the first 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 , 6.0, and 8.0 h. The samples withdrawn were diluted with fresh buffer, and the absorbance of samples was then read by using a UV spectrophotometer (Perkin Elmer Lambda XLS, USA) to determine the percentage of drug release. The samples were analyzed at a wavelength of 292 nm for NAR [5]. With the readings obtained, the percentage of drug release was calculated and the graph was plotted.

2.3 Microbiology test

The Kirby–Bauer test, which is also known as the disc diffusion test, was used to evaluate the antibiotic susceptibility of the four formulations, which include blank, gentamicin-and NAR-loaded hydrogel and hydrogel formulation loaded with gentamicin, and NAR along with the commercial preparation of gentamicin. The blank formulation was tested as a control. Agar plates were used for the current experiment where a sterile cotton swab containing Escherichia coli was used to swab the surface of agar and streak on the surface of agar of all the plates. The plates were streaked in one direction and it was rotated at 90° before streaking the plate again to obtain uniform bacterial growth. The rotation was repeated three times, completing a full rotation of 360°. All the plates were allowed to dry for 3–5 min. A measured quantity of prepared formulations was soaked into the sterile discs, except for the marketed product. The discs were placed on the agar using sterilized forceps. The discs were pressed gently to the agar using the sterilized tip of the forceps to ensure that the disc is attached to the agar surface and does fall off when the plate is inverted. To place the marketed product, a sterilized cork borer was used to punch a hole in the agar where the weighed quantity of the formulation was placed. The agar plates were then closed with the cover plate, sterilized on the surface, and incubated overnight at 37°C [36].

2.4 In vitro cytotoxicity assay

The HaCaT cells were cultured in DMEM, which was supplemented with FBS (10%) and penicillin–streptomycin (1%). Following the attachment of approximately 10,000 cells in each well, the cells were incubated with different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 µg/mL) of three different formulations, gentamicin-and NAR-loaded hydrogels and hydrogel formulation loaded with gentamicin and NAR, for 24 h at 37°C in a carbon dioxide (5%) incubator. An equivalent volume of the blank formulation was parallelly run to check the cytotoxicity of the blank formulation. Cells in the DMEM medium were kept as a negative control in the assay. For this experiment, each concentration was tested in triplicate. Following 72 h of incubation, the treatments were removed and the cells were again incubated in the DMEM media with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution for 4 h. Thereafter, following the removal of the media, the cells were treated with dimethyl sulfoxide to dissolve the formazan crystals. The formazan solutions in the wells were measured using a microplate reader (Tecan Infinite 200 pro, Switzerland) at 570 and 630 nm. The percentage of cell viability in the treated groups was calculated using the following formula [32]:

Percentage of cell viability = OD 570 − 630 treated cell OD 570 − 630 untreated cells × 100 % .

2.5 Statistical analysis

All experiments were performed in triplicate, and the results are presented as mean ± standard deviation. Statistical analysis was conducted using a one-way ANOVA to statistically evaluate the significance of differences between different groups. p < 0.05 is considered statistically significant.

3 Results and discussion

3.1 Selection of the mucoadhesive polymer

Aqueous dispersions of different polymers, such as HPMC, CMC, Carbopol 934, and Carbopol 940, were prepared as described and evaluated in terms of viscosity and texture by immersing a glass rod. A little of the formulation was placed on the fingertip and tested for its stickiness. The results are recorded in Table 2. From the table, it can be inferred that the polymers, such as HPMC, Carbopol 934, and Carbopol 940, have shown good adhesive properties. However, for CMC, there was no sign of adhesive property observed within the first three tested concentrations; hence, CMC was removed from our choice of mucoadhesive polymers. The other three polymers with good adhesive properties were further evaluated for the strength of mucoadhesiveness, where the last three concentrations of the three polymers were used to evaluate the mucoadhesive strength (data not shown). Based on the findings, HPMC has shown the strongest adhesive strength among the three (13–19 g with 0.4–0.6% HPMC, respectively). Hence, HPMC was chosen as the mucoadhesive polymer to be used in the preparation of in situ hydrogel formulation.

Table 2

Evaluation of viscosity and the texture of mucoadhesive polymers

Quantity	Polymers
Quantity	HPMC	CMC	Carbopol 934	Carbopol 940
0.1% w/v	Not sticky	Not sticky	Slightly sticky	Not sticky
0.2% w/v	Not sticky	Not sticky	Slightly sticky	Slightly sticky
0.3% w/v	Slightly sticky, not viscous	Not sticky	Sticky, adhered, and viscous	Sticky, adhered, and viscous
0.4% w/v	Slightly sticky, adhered, viscous	Not sticky	Sticky, adhered, and viscous	Sticky, adhered, and too viscous
0.5% w/v	Sticky, adhered, and viscous	—	Sticky, adhered, and too viscous	Sticky, adhered, and too viscous
0.6% w/v	Sticky, adhered, and viscous	—	Sticky, adhered, and too viscous	Sticky, adhered, and too viscous

3.2 Selection of the thermo-responsive polymer

Pluronic F-127 and Pluronic F-68 were used as thermo-sensitive polymers and they were evaluated by determining the temperature gelling point using the test tube inversion method. Aqueous concentrations of 15.0, 18.5, 19.0, 19.5, and 20.0% of Pluronic F-127 [37] and 9.0, 10.0, and 11.0% of Pluronic F-68 [38] were tested based on the available literature data. Additionally, the combination of both the polymers containing 10% Pluronic F-127 and 20% Pluronic F-68, and another combination containing 10% Pluronic F-127 and 15% Pluronic F-68 were tested separately.

The temperature of the chronic wound environment is in the range of 31–35°C [39], which is targeted for the ideal gelling point of the formulations. It was observed that Pluronic F-127 reached its gelling state within the chronic wound temperature at concentrations of 19.0, 19.5, and 20.0%. The formulations of Pluronic F-68 polymer and the formulations containing the combination of Pluronic F-127 and Pluronic F-68 were in the liquid state up to the tested range of temperature (40°C), which is above the range of the chronic wound temperature. Hence, Pluronic F-127 was chosen as the thermo-responsive polymer to prepare the in situ hydrogel formulation.

3.3 Hyaluronic acid as the pH-responsive polymer

pH is one of the most important properties where a wound environment with an acidic environment helps in promoting wound healing [5]. In an alkaline environment, acidic polymers could become negatively charged after deprotonation and exhibit swelling properties. As the pH of the chronic wound is alkaline (pH 7.15–8.90), therefore, the ideal pH of the formulation should be acidic in order to display swelling properties and forms a gel-like structure, which will promote a sustained release property of the entrapped drugs to make it useful for topical application in wounds. In this case, hyaluronic acid was chosen as it is classified as a natural pH-sensitive polymer that swells at basic pH. Based on research done by Kim et al., it was found that hyaluronic acid attained the highest swelling ability when it is exposed to an environment with a pH of 7.0–8.0. In addition, it helps in wound healing and it is widely used in wound dressing because of its positive role in preserving tissue integrity and promoting cell adhesion during the inflammation process of wound healing [40]. However, considering the incorporation of other polymers, two concentrations (0.05 and 0.1%) were selected for the optimization process.

3.4 Optimization of the pH-responsive polymer, thermo-responsive polymer, and mucoadhesive polymer

3.4.1 Effect of independent variables on the gelling temperature

Gelling temperature is an extremely important variable in in situ hydrogels for topical application as it is expected to turn the sol to a gel-like state when in contact with the wound environment. If it fails, the formulation will not be retained at the site of action and its effectiveness decreases [41]. The statistical results on the effect of three independent variables, A (HPMC), B (Pluronic F-127), and C (hyaluronic acid), on the dependent variables (gelling temperature) for the stimuli-responsive hydrogel are presented in Table 3. The statistical significance of the influence of three independent variables on the gelling temperature is represented by the p value (0.0003) in Table 3; it can be said that A, B, and C are significant model terms. Thus, it can be inferred that the model terms (A, B, and C) have a significant effect on the gelling temperature of the stimuli-responsive hydrogel. The F-value for the lack of fit of 0.81 represents that the lack of fit is not significant relative to the pure error, which is further confirmed by the p value. It is good to have a non-significant lack of fit as the expectation is for the model to be fit. Furthermore, the predicted R² (0.88) and adjusted R² (0.97) values are in close agreement, as the difference is less than 0.2. In this model, the adequate precision value of 23.3, which measures the signal/noise ratio, implied an adequate signal as the signal/noise ratio is more than 4. Henceforth, this model could be used to navigate the design space.

Table 3

ANOVA data for the gelling temperature and viscosity following the quadratic model

Source	Response 1: Gelling temperature				Response 2: Viscosity
Source	Sum of squares	Mean square	F-value	p-value	Sum of squares	Mean square	F-value	p-value
Model	58.89	6.54	45.18	0.0003 (significant)	1.035 × 10⁵	11497.04	49.27	0.0002 (significant)
A-HPMC	2.76	2.76	19.07	0.0072	18050.00	18050.00	77.36	0.0003
B-Pluronic F-127	51.01	51.01	352.16	<0.0001	8450.00	8450.00	36.21	0.0018
C-Hyaluronic acid	3.78	3.78	26.11	0.0037	64800.00	64800.00	277.71	<0.0001
AB	0.16	0.16	1.10	0.3414	100.00	100.00	0.43	0.5416
AC	0.06	0.06	0.43	0.5403	3600.00	3600.00	15.43	0.0111
BC	0.00	0.00	0.00	1.0000	400.00	400.00	1.71	0.2474
A²	0.24	0.24	1.65	0.2556	125.64	125.64	0.54	0.4960
B²	0.06	0.06	0.43	0.5431	2464.10	2464.10	10.56	0.0227
C²	0.73	0.73	5.07	0.0742	6156.41	6156.41	26.38	0.0037
Residual	0.72	0.14			1166.67	233.33
Lack of fit	0.40	0.13	0.81	0.5933 (not significant)	900.00	300.00	2.25	0.3224 (sot significant)

Additionally, the interaction of three independent variables and their effect on the gelling temperature of stimuli-responsive hydrogel is represented in equation (2), where the coefficients for A, B, and C are −0.5875, −2.52, and −0.6875, respectively. The negative coefficient represented a decrease in the gelling temperature with increasing concentrations of HPMC, Pluronic F-127, and hyaluronic acid in the formulations and, further, the effect of all three polymers is significant, which is indicated by the p value:

(2) Y 1 = + 34.43 − 0.5875 ⁎ A − 2.52 ⁎ B − 0.6875 ⁎ C − 0.2542 ⁎ A 2 − 0.2000 ⁎ A ⁎ B − 0.1250 ⁎ A ⁎ C − 0.1292 ⁎ B 2 + 0.0000 ⁎ B ⁎ C + 0.4458 ⁎ C 2 .

In equation (2), the coefficient value of B is much higher than the coefficient values of A and C, which presents the highest effect of B when compared to A and C on the gelling temperature. This is further confirmed by the perturbation plot (Figure 2a), contour plot (Figure 3a and d), surface plot (Figure 3b) on the effect of independent variables on gelling temperature. In the perturbation plot (Figure 2a), a sharp decline of the gelling temperature with an increasing concentration of B in the formulation represented the higher effect of B when compared to A and C on the gelling temperature. In addition, the declined slope with the increasing concentrations of A, B or C variables could be correlated to the negative coefficients of A, B and C in the equation (2). Further, a similar effect is reflected in contour plots (Figure 3a and d) and surface plots (Figure 3b), where higher changes in the gelling temperature (maximum color changes) are represented through the Pluronic F-127 axis. The closeness of experimental and predicted values is represented in Figure 2c.

Figure 2

Perturbation plots (a and b) and linear correlation plots between the actual and predicted values (c and d) for the gelling temperature and viscosity.

Figure 3

Contour plots (a and c) and three-dimensional surface response plots (b and d) showing the effect of incorporated polymers in in situ hydrogels on the gelling temperature and viscosity.

3.4.2 Effect of independent variables on the viscosity of the stimuli-responsive hydrogel

Viscosity is another important property that should possess the desired consistency to ensure complete coverage of the wound surface by the formulation and aid in sustained drug release from the drug-loaded formulations [42]. The statistical outcome of the interaction of three independent variables at the three levels in viscosity of the stimuli-responsive hydrogel is represented in Table 3. The statistical significance of the influence of three independent variables, A (HPMC), B (Pluronic F-127), and C (hyaluronic acid) on the viscosity is presented by the p value ( <0.05); it can be inferred that the model terms A, B, C, AC, B², and C² are the significant model terms. The three polymers have shown a significant effect on the viscosity of the formulations. The interaction of the three independent variables and their effect on the viscosity of stimuli-responsive hydrogel is represented in equation (3), where A, B, and C have the positive coefficients, which indicate an increase in viscosity with increasing concentration of all three polymers in the formulations. The model F value, which represents the significance of the model F-value for the lack of fit of 2.25, shows that the lack of fit is not significant relative to the pure error, which is confirmed by the p value. It is good to have a non-significant lack of fit as the expectation is for the model to be fit. Furthermore, predicted R² (0.8567) and adjusted R² (0.9688) values are in close agreement with a difference of less than 0.2:

(3) Y3 = + 233 .3 + 47 .50⁎A + 32 .50⁎B + 90 .00⁎C + 5 .83⁎A 2 − 5 .00⁎A⁎B - 30 .00⁎A⁎C + 25 .83⁎B 2 − 10 .00⁎B⁎C + 40 .83⁎C 2 .

The higher coefficient value of C than those of A and B in equation (3) indicated the higher effect of C when compared to A and B on enhancing the viscosity of the stimuli-responsive hydrogel. The findings are in agreement with the perturbation plot (Figure 2b), contour plot (Figure 3c and d), and surface plot (Figure 3d) on the effect of independent variables on the viscosity of the formulation. The increasing slope in the perturbation plot (Figure 2b) is in agreement with the positive coefficient for model terms A, B, and C in equation (3). Further, a higher slope with an increasing concentration of C in the formulation represented the higher effects of C when compared to A and B on the viscosity of the formulations, which is in agreement with the highest coefficient of C in equation (3). Furthermore, a similar effect is reflected in the contour plot (Figure 3c and d) and surface plots (Figure 3d), where higher changes in viscosity (maximum color changes) are evident through the hyaluronic acid axis. The closeness of experimental and predicted values of viscosity is evident in Figure 2d.

3.5 Preparation of drug-loaded optimized in situ hydrogel formulations

Four optimized formulations were prepared, which included blank stimuli-responsive in situ hydrogels and three drug-loaded formulations containing only gentamicin (0.3%), only NAR (0.5%), and the combination of gentamicin (0.3%) and NAR (0.5%). Based on the optimization process by the Design Experiment, the concentrations of HPMC, Pluronic F-127, and hyaluronic acid were fixed. For all the preparations, 0.51% of HPMC, 18.8% of Pluronic F-127, and 0.08% of hyaluronic acid were used. The blank formulation acts as a control throughout the experiment.

3.6 TGA

TGA was performed to determine the temperature at which the substance degrades. The range of temperature exposed for each substance varies based on the sources for each ingredient. Based on graph I in Figure 4, the TGA of gentamicin sulfate, there were two significant weight losses that were observed: one was nearly below 100°C and the other one was at approximately 280 to 400°C. Up to 600°C, gentamicin sulfate was found to have 31.4% weight remaining and 10% of the weight loss was achieved at 98°C, which was the first weight loss as shown in Graph I. Looking at graph II, the TGA of NAR, there was one weight-loss event that occurred at approximately 330°C, which is further supported by the research conducted by Fuster et al. [27]. Also, a 10% weight loss of NAR was observed at 338°C, which was nearly the temperature at which it starts to degrade. It still had the remaining 25.3% until exposure to a temperature of 800°C. Based on graph III, the TGA of hyaluronic acid, two stages of weight loss were observed where the first one was below 100 to approximately 150°C and the other one was at nearly 280°C. The first weight loss of 10% at 95.88°C might be related to the process by which the structural and free water evaporates and the second weight loss was the actual degradation of the hyaluronic acid itself [43]. There was 26.2% remaining after being exposed to 800°C, and this might be due to the excess amount of hyaluronic acid that was used for the analysis. Based on graph IV, the TGA of Pluronic F-127, there was a single-step declination in the weight loss, which was approximately between 350 and 400°C and nearly the range reported by Nguyen et al. [44] and 10% of weight loss was found to be within the range that was at 350.05°C. There was a remaining 1.22% with exposure up to 500°C. Based on graph V, the TGA of HPMC, there were two weight loss stages observed: one was nearly 60°C and another temperature within the range of 340–400°C, as reported by Yin et al. where the range of temperatures was nearly the same [45]. The presence of two functional groups in HPMC might be the reason that contributes to the two stages of degradation. From the findings, the idea of degradation temperatures of the individual pure compounds was obtained, which had been employed in the DSC evaluation.

Figure 4

TGA thermograms of gentamicin sulfate (I), NAR (II), hyaluronic acid (III), Pluronic F-127 (IV), and HPMC (V).

3.7 DSC analysis

DSC measures the total heat flow, using temperatures in and out of material and it was mainly carried out in this study to analyze the interaction between the therapeutic molecules, gentamicin, and NAR, with the other three polymers, HPMC, Pluronic F-127, and hyaluronic acid. Based on the DSC of gentamicin in Figure 5-II, the melting point was observed at a peak of 159.18°C within the range of 148.68–176.64°C and an enthalpy of −979.16 mJ. Also, a sharp peak was noticed at 247.81°C with an enthalpy of 124.42 mJ. Based on the DSC of NAR, the melting point was observed at a sharp peak of 251.85°C, within the range of 250.0–256.71°C and an enthalpy of −1752.68 mJ. Based on the DSC of hyaluronic acid, the melting point was observed in the range of 162.37–177.95°C with a peak at 163.73°C and an enthalpy of −1805.88 mJ; another sharp peak was observed at 232.85°C with an enthalpy of 1687.14 mJ. Based on the DSC of HPMC, the melting point was observed in the range of 133.88–173.91°C with a peak at 143.59°C and an enthalpy of −447.75 mJ. Based on the DSC of Pluronic F-127, the melting point was noticed in the range of 51.51–62.82°C with a peak at 56.59°C and an enthalpy of −1813.61 mJ. Also, a sharp peak was noticed at 357.89°C with an enthalpy of −130.05 mJ. Missing peaks and reduction in the temperature as compared to the reference indicate the interaction between the excipient and the therapeutic materials [46] and it was determined by comparing the combination graph with the other individual graphs. Based on the DSC of the combination, three peaks were observed. The first peak was observed at 35.26°C with an enthalpy of −8.39 mJ, the second peak was found at 47.9°C with an enthalpy of −45.26 mJ, and the third peak was observed at 125.44°C with an enthalpy of −587.64 mJ. The second peak temperature from the combination graph is found to be nearby the melting point of Pluronic 127; however, there was an increase in the enthalpy from −1813.61 to −45.26 mJ. The third peak and its range from the combination graph were also found to be nearby to the melting point peak and the range of HPMC. There was a decrease in the enthalpy from −447.75 to −587.64 mJ. A peak was observed in the combination graph (Figure 5-I) at approximately 250°C, nearby the peak of the melting point of NAR with a negative enthalpy. Similarly, a peak was observed at approximately 230°C in the combination graph with a positive enthalpy, which correlates with the peak observed from DSC of hyaluronic acid. However, there were differences in the combination graph where it had an additional peak at a lower temperature, which did not correlate with other substances. Also, the peak melting point that was observed in the DSC thermogram of gentamicin and of hyaluronic acid was not seen in the DSC thermogram. This shows that there is an interaction between the excipients and the therapeutic agents.

Figure 5

DSC thermograms of gentamicin sulfate (II), NAR (III), hyaluronic acid (IV), HPMC (V), Pluronic F-127 (VI), and the combination thereof (I).

3.8 FTIR spectroscopy analysis

FTIR analysis was conducted to evaluate the compatibility of the substances to be used in the preparation of in situ hydrogel formulation by obtaining the infrared spectrum of each substance and comparing it with the combination. Based on the spectrum of gentamicin in Figure 6, there were bands at 1622.32, 1519.89, and 1384.62 cm^−1, which indicates the amide bonds; to be specific, amide I, amide II, and amide III, respectively [28]. There was also a peak observed which is at 617.98 cm⁻¹, which indicates the presence of sulfur, which is in the form of S–O stretching and vibration [47]. In the NAR spectrum, bands were observed at peaks of 3288.02 and 3117.09 cm⁻¹, which indicates the O–H stretching, and a peak was observed at 1606.72 cm⁻¹, which indicates the C═O stretching [48]. Based on the hyaluronic acid spectrum, peaks were observed at 1,720 and 1,253 cm⁻¹, which indicates the stretching region of the protonated COOH group. There was a band with a peak at 3509.03 cm⁻¹, which indicates the stretching regions of the NH and OH groups. A stretching vibration region of the CH group was detected at the band with a peak at 2915.70 cm⁻¹. There was also a sharp peak observed at 1,635 cm⁻¹, which indicates the amide carbonyl group, and the stretching of the functional group of COO⁻ was also noticed at a band of 1,418 cm⁻¹ [49]. Based on the spectrum of HPMC, OH stretching was observed by an absorption band with a peak at 3444.99 cm⁻¹. Also, the stretching vibrations of bonds C–H and C–O were detected by the bands with a peak at 2901.41 and 1054.72 cm⁻¹, respectively. A band was present at 1376.25 cm^−1, which indicates that there are bending vibrations of the OH functional groups [50]. In the Pluronic F-127 spectrum, there was a stretching vibration region of C–H detected by the presence of the absorption peak at 2884.36 cm⁻¹. Also, bending vibrations of the OH functional groups were detected by the presence of a band at 1341.98 cm⁻¹ and there were stretching vibrations of the C–O–C functional group by the absorption peak at 1098.87 cm⁻¹ [51]. Based on the FTIR spectrum of the combination, the presence of amide bonds at 1739.80 and 1588.57 cm⁻¹ and a slight shift of the peaks to 1622.32 and 1519.89 cm indicate the presence of gentamicin sulfate. This might be attributed to the possible formation of H-bonds between the molecules. There was a band at 763.01 cm⁻¹ indicating the stretching and vibration of S–O, which is present in gentamicin sulfate. The OH stretching was found by the presence of bands at 3174.62 and 3108.44 cm⁻¹. Also, bands were present at 1086.90 and 1103.67 cm⁻¹ indicating the stretching and vibration of the functional group C–O–C. The bending vibration of –OH was detected at the absorption peak of 1377.39 and 1408.74 cm⁻¹. Also, the protonated COOH group was detected by the absorption peak at 1739.98 and 1281.67 cm⁻¹.

Figure 6

FTIR spectra of gentamicin sulfate (II), NAR (III), hyaluronic acid (IV), HPMC (V), Pluronic F-127 (VI), and the combination thereof (I).

3.9 Mucoadhesive strength of the finalized optimized formulations

The mucoadhesive property of the formulation helps in establishing the ability of a topical formulation to retain at the site of action when applied to a particular area [52]. The mucoadhesive property was determined by evaluating the strength of the mucoadhesive and the force of adhesion, which were calculated from the strength of the mucoadhesive. The force of adhesion of the blank and three drug-loaded formulations was calculated from the following formula [5]:

force of adhesion ( N ) = M ucoadhesive strength 1 , 000 × 9 . 8 kg m / s 2 .

As presented in Figure 7a, it can be inferred that the difference in the force of adhesion between different formulations is statistically not significant. HPMC possesses a good strength of adhesion and moderate swelling property, which aids in protecting the surface of the wound [53]. The gel was applied topically at the body temperature; it shows the ability of the Pluronic F-127 to form a more stable form, into the gellified state, which helps in retaining within the applied area. Hyaluronic acid is a natural polyacid polymer, which swells at a high pH environment and this helps to form a more stable formulation to retain at the site of action. This is further proven in the results displayed by the Box–Behnken statistical design where hyaluronic acid has shown a higher effect on the adhesive property. As the differences between the mucoadhesive strength and the force of adhesion of the four formulations were not significant, it can be inferred that the therapeutic agents did not contribute to the adhesive property of the drug-loaded formulations.

Figure 7

Presentation of the (a) mucoadhesive strength and force of adhesion of the optimized blank and drug-loaded in situ hydrogels and (b) in vitro release profile of NAR from the drug-loaded formulations in PBS (pH 7.4) at 33°C. Values are expressed as mean ± SD (n = 3).

3.10 Viscosity of formulations

Viscosity is an extremely important property in a topical formulation that helps in enhancing the residence time of the formulation thereby providing a sustained release property for the formulation. Most importantly, a targeted viscosity is desired to produce a formulation that is smooth and has higher retention at the site of application [5]. Based on the results displayed in Table 4, there is no significant difference between the obtained results on viscosity, when the drug-loaded formulations are compared with the blank formulation. This suggests that the addition of drugs into the formulation did not interfere with the viscosity of the final formulation. The viscosity readings could be related to the results of our mucoadhesive strength. It is well understood that a higher viscosity of a formulation will contribute to better adhesive properties [54].

Table 4

Mucoadhesive strength, viscosity, gelling temperature, and area of coverage of a single spray of the optimized in situ hydrogel formulations

Formulation	Mucoadhesive strength* (g)	Force of adhesion* (N)	Viscosity* (cP)	Gelling temperature (°C)	Spray coverage area* (cm²)
Drug-free formulation	29.6 ± 0.6	0.29 ± 0.01	174 ± 3	30	18.1 ± 0.8
Gentamicin-loaded	30.3 ± 0.6	0.30 ± 0.01	178 ± 4	33	16.8 ± 1.5
NAR-loaded	30.3 ± 0.6	0.30 ± 0.01	176 ± 4	32	17.4 ± 0.7
Combination	31.3 ± 0.6	0.31 ± 0.01	184 ± 4	31	18.9 ± 1.2

*Values are expressed as mean ± SD (n = 3).

3.11 The temperature of the gelling point

Formulating a topical formulation with thermo-responsive polymers shows the temperature of the gelling point. It is an extremely important factor that helps ensure the conversion of sol-to-gel when in contact with the wound environment. The evaluated gelling temperatures of the four formulations are tabulated in Table 4. It can be observed that the drug-loaded formulations reached a gel-like structure at a temperature, which was in the range of a chronic wound temperature (31–35°C). The blank formulation was gellified at a temperature that was not within the range; however, it was not significant as the difference was only by a decrement of 1°C. Hence, the concentration of Pluronic F-127 of 18.8% is concluded to be suitable to be used as a thermosensitive polymer in the preparation of in situ hydrogels for chronic wound application.

3.12 Spray dimension test

The purpose of carrying out the spray dimension test was to determine the coverage area of the formulation, where it can display the ability of the formulation to spread its coverage to a certain area. A sufficient coverage area is an ideal property of an in situ hydrogel formulation for topical application on chronic wounds to ensure coverage of the wound area using a minimum spray. The four optimized formulations were tested for the spray dimension and the area of coverage was calculated and is presented in Table 4. The drug-loaded formulations as well as the blank formulation had a wider area of coverage with just a single spray, indicating that these are able to cover a large area of the wound, assuming to spread a certain amount of antimicrobial agents to the covered area.

3.13 In vitro drug release test using the Franz diffusion cell method

A drug release test is a vital property in characterizing the performance of a particular dosage form under standardized in vitro conditions to provide a better understanding of the in vivo aspect of the particular product [52]. In this case, the Franz diffusion cell method was used to measure the in vitro drug release of the three drug-loaded formulations. As observed from Figure 7b, the formulations had a fast release, starting from the first half an hour till the second hour. This could be considered as an initial burst release of the entrapped drugs, which might help in establishing antimicrobial efficacy besides avoiding the potential antimicrobial resistance. The formulations possess the sustained release property of NAR, which might help to supply the drug from the gel matrix to the wound environment for a longer duration. The release property of gentamicin could be expected similar to or even better than NAR because of the aqueous soluble property of gentamicin. However, the presence of the polymeric matrix would help prolong the release of the drugs from the gel matrix. Furthermore, the release pattern of NAR from both formulations is quite comparable, indicating that the presence of two drugs does not interfere with the release pattern. The obtained results indicate that the combination formulation would be able to release the antimicrobial contents slowly to exhibit a better wound healing property due to the presence of two components.

3.14 Microbiological assay

The disc diffusion test was used to evaluate the antibiotic susceptibility of the blank and three drug-loaded formulations and they were compared with a commercial product of 0.3% gentamicin cream. The results of the zone of inhibition on the agar plate are recorded in Figure 8, where the formulation containing both gentamicin and NAR had a larger zone of inhibition of 2.03 ± 0.12 cm as compared to the other formulations with individual drug-loaded formulations and also the 0.3% commercial gentamicin. Significant changes in the zone of inhibition were observed in gentamicin-loaded, co-loaded, and commercial formulations while compared to only NAR-loaded formulation (p < 0.05). Although the diameter of thezone of inhibition for the formulation containing gentamicin and NAR was superior than the zone of inhibition found with only gentamicin-loaded formulation, the difference is statistically insignificant. Furthermore, the difference in the diameter of the zone of inhibition was found to be statistically significant when compared to the co-delivered formulation and commercial formulation (p < 0.05). This might be due to the synergistic role of the two added compounds in the co-delivered hydrogel. The role of gentamicin and NAR in wound healing has already been shown in the literature [55,56], where the antimicrobial activity of the two might be one of the underlying mechanisms of healing wounds.

Figure 8

Comparative presentation of antimicrobial activities of the optimized blank and drug-loaded in situ hydrogels and commercial formulation while incubated at 37°C for 24 h; (a) tabular presentation of comparative findings of zone of inhibitions, values are expressed as mean ± SD (n = 3), (b) and (c) are presentation of zone of inhibitions in discs. *Significant changes in the zone of inhibition were observed in gentamicin-loaded, co-loaded, and commercial formulations when compared to only NAR-loaded formulation, whereas #co-loaded formulation showed a significant increase in the zone of inhibition when compared to the commercial formulation (p < 0.05).

3.15 Evaluation of cytotoxic effects

In due course of evaluating the cytotoxic potential of the blank formulation and the drug-loaded formulations, we have used HaCaT cells. The concentration range selected for evaluating the cytotoxicity of the formulation ranged from 1.56 to 100.0 µg/mL, where the blank formulation was incorporated in the evaluation at the same concentration. The determined IC₅₀ values of the treated blank formulation and the drug incorporated (individual gentamicin, NAR, and combination thereof) showed good results where all the formulations showed values >100 µg/mL. Therefore, the blank formulation and drug(s) incorporated formulations could be referred that are relatively safe for the experimental HaCaT cells. Thus, it could be inferred that the incorporation of drugs into the compositions of in situ hydrogel would not interfere with the toxicity of the formulations. Thus, the fabricated formulations would be optimum for topical application to the wound environment [32].

4 Conclusions

To summarize, an optimized formulation containing stimuli (pH- and thermo-) responsive polymers was formulated using the Box–Behnken model where the optimized gelling temperature and viscosity were obtained with varying percentages of hyaluronic acid, Pluronic F-127, and HPMC. The optimized formulations were found to be changed into a gel-like state at a temperature within the range of temperature of chronic wounds, displaying its ability to swell. The viscosities of the formulations are desirable to allow spray over the wound area with the display of good adhesive properties. Overall, from the findings of the designed research, it can be said that the combination of two components (NAR and gentamicin) in a stimuli-sensitive hydrogel platform would be useful in the treatment of topical wounds where there are no physical or chemical interactions between the excipients and actives. Furthermore, the sustained release profile of the hydrophobic component in the formulation suggests prolonged action, where no interaction of the other component is expected to have a similar release profile because the drugs are entrapped in the same matrix. The superior antimicrobial properties of the combined formulation might be due to the sustained release of the two components from the formulation, which is significant when compared to the commercial formulation of gentamicin or similar formulation containing NAR, which is ideal for topical administration in the wound environment. Furthermore, the stimuli-sensitive hydrogel formulations showed potential for topical application as these showed lower cytotoxicity. Hence, the pH- and thermo-sensitive hydrogel containing gentamicin and NAR would give new insights into chronic wound healing as it displayed the potential of being an effective treatment, with the hope of being able to improve the quality of life in patients with a chronic wound in future. However, more studies are indeed necessary to prove its efficacy and toxicity in specific animal models.

bapi.gorain@bitmesra.ac.in

# Current address: Department of Pharmaceutical Sciences, Central University of Haryana, SSH 17, Jant, Haryana 123031, India.

Acknowledgements

The authors would like to thank Taylor’s University, Malaysia, for providing the research facilities.

Funding information: The Deanship of Scientific Research (DSR), King Abdulaziz University (KAU), Jeddah, Saudi Arabia has funded this Project, under grant no. KEP-MSc: 11-166-1443.
Author contributions: B.G. – conceptualization, methodology, formal analysis, writing – review and editing, visualization, supervision; M.P. – methodology, data curation, writing – original draft preparation; H.C. – methodology, data curation, formal analysis, writing – original draft preparation; S.M. – methodology, investigation, visualization, funding acquisition; T.T. – data curation, writing – original draft preparation; Y.K.H. – data curation, writing – original draft preparation; M.H.A. – data curation, writing – original draft preparation; L.W.M. – data curation, writing – original draft preparation.
Conflict of interest: The authors declare no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data generated or analyzed during this study are included in this published article.

References

[1] Gorain B, Pandey M, Leng NH, Yan CW, Nie KW, Kaur SJ, et al. Advanced drug delivery systems containing herbal components for wound healing. Int J Pharm. 2022 Apr;617:121617.10.1016/j.ijpharm.2022.121617Search in Google Scholar PubMed

[2] Jeckson TA, Neo YP, Sisinthy SP, Gorain B. Delivery of therapeutics from layer-by-layer electrospun nanofiber matrix for wound healing: An update. J Pharm Sci. 2021 Oct [cited 2020 Oct 17];110(2):635–53. https://linkinghub.elsevier.com/retrieve/pii/S0022354920305888.10.1016/j.xphs.2020.10.003Search in Google Scholar PubMed

[3] Järbrink K, Ni G, Sönnergren H, Schmidtchen A, Pang C, Bajpai R, et al. Prevalence and incidence of chronic wounds and related complications: A protocol for a systematic review. Syst Rev. 2016 Sep [cited 2021 May 1];5(1):152. http://systematicreviewsjournal.biomedcentral.com/articles/10.1186/s13643-016-0329-y.10.1186/s13643-016-0329-ySearch in Google Scholar PubMed PubMed Central

[4] Power G, Moore Z, O’Connor T. Measurement of pH, exudate composition and temperature in wound healing: A systematic review. J Wound Care. 2017 Jul [cited 2021 May 1];26(7):381–97. 10.12968/jowc.2017.26.7.381 Search in Google Scholar PubMed

[5] Akrawi SH, Gorain B, Nair AB, Choudhury H, Pandey M, Shah JN, et al. Development and optimization of naringenin-loaded chitosan-coated nanoemulsion for topical therapy in wound healing. Pharmaceutics. 2020 Sep [cited 2020 Sep 26];12(9):893. https://www.mdpi.com/1999-4923/12/9/893.10.3390/pharmaceutics12090893Search in Google Scholar PubMed PubMed Central

[6] Choudhury H, Pandey M, Lim YQ, Low CY, Lee CT, Marilyn TCL, et al. Silver nanoparticles: Advanced and promising technology in diabetic wound therapy. Mater Sci Eng C. 2020 Jul;112:110925.10.1016/j.msec.2020.110925Search in Google Scholar PubMed

[7] Yeo E, Yew Chieng CJ, Choudhury H, Pandey M, Gorain B. Tocotrienols-rich naringenin nanoemulgel for the management of diabetic wound: Fabrication, characterization and comparative in vitro evaluations. Curr Res Pharmacol Drug Discov. 2021 Jan [cited 2021 Mar 29];2:100019. https://linkinghub.elsevier.com/retrieve/pii/S2590257121000067.10.1016/j.crphar.2021.100019Search in Google Scholar PubMed PubMed Central

[8] Bektas N, Şenel B, Yenilmez E, Özatik O, Arslan R. Evaluation of wound healing effect of chitosan-based gel formulation containing vitexin. Saudi Pharm J. 2020 Jan;28(1):87–94.10.1016/j.jsps.2019.11.008Search in Google Scholar PubMed PubMed Central

[9] Shende P, Gupta H. Formulation and comparative characterization of nanoparticles of curcumin using natural, synthetic and semi-synthetic polymers for wound healing. Life Sci. 2020 Jul;253:117588.10.1016/j.lfs.2020.117588Search in Google Scholar PubMed

[10] Hoare TR, Kohane DS. Hydrogels in drug delivery: Progress and challenges. Polymer (Guildf). 2008 Apr;49(8):1993–2007.10.1016/j.polymer.2008.01.027Search in Google Scholar

[11] Dai T, Tanaka M, Huang YY, Hamblin MR. Chitosan preparations for wounds and burns: Antimicrobial and wound-healing effects. Expert Rev Anti Infect Ther. 2011 Jul [cited 2020 Aug 30];9(7):857–79. https:/pmc/articles/PMC3188448/? report = abstract.10.1586/eri.11.59Search in Google Scholar PubMed PubMed Central

[12] Wu J, Zheng Y, Wen X, Lin Q, Chen X, Wu Z. Silver nanoparticle/bacterial cellulose gel membranes for antibacterial wound dressing: investigation in vitro and in vivo. Biomed Mater. 2014 Apr;9(3):035005.10.1088/1748-6041/9/3/035005Search in Google Scholar PubMed

[13] Lu Y, Li H, Wang J, Yao M, Peng Y, Liu T, et al. Engineering Bacteria-Activated Multifunctionalized Hydrogel for Promoting Diabetic Wound Healing. Adv Funct Mater. 2021;31(48):2105749.10.1002/adfm.202105749Search in Google Scholar

[14] Zheng M, Wang X, Yue O, Hou M, Zhang H, Beyer S, et al. Skin-inspired gelatin-based flexible bio-electronic hydrogel for wound healing promotion and motion sensing. Biomaterials. 2021;276:121026.10.1016/j.biomaterials.2021.121026Search in Google Scholar PubMed

[15] Zhang X, Yao D, Zhao W, Zhang R, Yu B, Ma G, et al. Engineering platelet-rich plasma based dual-network hydrogel as a bioactive wound dressing with potential clinical translational value. Adv Funct Mater. 2021;31(8):2009258.10.1002/adfm.202009258Search in Google Scholar

[16] Hu L, Wan Y, Zhang Q, Serpe MJ. Harnessing the power of stimuli‐responsive polymers for actuation. Adv Funct Mater. 2020 Jan [cited 2021 May 2];30(2):1903471. 10.1002/adfm.201903471 Search in Google Scholar

[17] Municoy S, Álvarez Echazú MI, Antezana PE, Galdopórpora JM, Olivetti C, Mebert AM, et al. Stimuli-responsive materials for tissue engineering and drug delivery. Int J Mol Sci. 2020 Jul [cited 2021 May 2];21(13):4724. https://pubmed.ncbi.nlm.nih.gov/32630690/.10.3390/ijms21134724Search in Google Scholar PubMed PubMed Central

[18] Hsin YK, Thangarajoo T, Choudhury H, Pandey M, Meng LW, Gorain B. Stimuli-responsive in situ spray gel of miconazole nitrate for vaginal candidiasis. J Pharm Sci. 2023;112(2):562–72.10.1016/j.xphs.2022.09.002Search in Google Scholar PubMed

[19] Wu J, Su ZG, Ma GH. A thermo- and pH-sensitive hydrogel composed of quaternized chitosan/glycerophosphate. Int J Pharm. 2006 Jun;315(1–2):1–11.10.1016/j.ijpharm.2006.01.045Search in Google Scholar PubMed

[20] Laurano R, Boffito M, Abrami M, Grassi M, Zoso A, Chiono V, et al. Dual stimuli-responsive polyurethane-based hydrogels as smart drug delivery carriers for the advanced treatment of chronic skin wounds. Bioact Mater. 2021 Feb;6(9):3013–24.10.1016/j.bioactmat.2021.01.003Search in Google Scholar PubMed PubMed Central

[21] Kim AR, Lee SL, Park SN. Properties and in vitro drug release of pH- and temperature-sensitive double cross-linked interpenetrating polymer network hydrogels based on hyaluronic acid/poly (N-isopropylacrylamide) for transdermal delivery of luteolin. Int J Biol Macromol. 2018 Oct;118:731–40.10.1016/j.ijbiomac.2018.06.061Search in Google Scholar PubMed

[22] Gemeinder JL, Barros NR, Pegorin GS, Singulani JD, Borges FA, Arco MC, et al. Gentamicin encapsulated within a biopolymer for the treatment of Staphylococcus aureus and Escherichia coli infected skin ulcers. J Biomater Sci Polym Ed. 2021 [cited 2021 May 2];32(1):93–111. 10.1080/09205063.2020.1817667 Search in Google Scholar PubMed

[23] Varga M, Sixta B, Bem R, Matia I, Jirkovska A, Adamec M. Application of gentamicin-collagen sponge shortened wound healing time after minor amputations in diabetic patients – A prospective, randomised trial. Arch Med Sci. 2014 [cited 2021 May 2];10(2):283–7. https://pubmed.ncbi.nlm.nih.gov/24904661/.10.5114/aoms.2014.42580Search in Google Scholar PubMed PubMed Central

[24] Tripathi A, Awasthi H, Rokaya DB, Srivastava D, Srivastava V. Antimicrobial and wound healing potential of dietary flavonoid naringenin. Nat Prod J. 2019 Feb [cited 2021 May 2];9(1):61–8. http://www.eurekaselect.com/164275/article.10.2174/2210315508666180802104630Search in Google Scholar

[25] Halligan SC, Dalton MB, Murray KA, Dong Y, Wang W, Lyons JG, et al. Synthesis, characterisation and phase transition behaviour of temperature-responsive physically crosslinked poly (N-vinylcaprolactam) based polymers for biomedical applications. Mater Sci Eng C. 2017 Oct;79:130–9.10.1016/j.msec.2017.03.241Search in Google Scholar PubMed

[26] Kumbhar SA, Kokare CR, Shrivastava B, Gorain B, Choudhury H. Preparation, characterization, and optimization of asenapine maleate mucoadhesive nanoemulsion using Box-Behnken design: In vitro and in vivo studies for brain targeting. Int J Pharm. 2020 Jun;586:119499.10.1016/j.ijpharm.2020.119499Search in Google Scholar PubMed

[27] Fuster MG, Carissimi G, Montalbán MG, Víllora G. Improving anticancer therapy with naringenin-loaded silk fibroin nanoparticles. Nanomaterials. 2020 Apr [cited 2022 May 30];10(4):718. https://pubmed.ncbi.nlm.nih.gov/32290154/.10.3390/nano10040718Search in Google Scholar PubMed PubMed Central

[28] Dwivedi C, Pandey H, Pandey AC, Patil S, Ramteke PW, Laux P, et al. In vivo biocompatibility of electrospun biodegradable dual carrier (Antibiotic + growth factor) in a mouse model—implications for rapid wound healing. Pharmaceutics. 2019 Apr [cited 2021 May 2];11(4):180. https://pubmed.ncbi.nlm.nih.gov/31013995/.10.3390/pharmaceutics11040180Search in Google Scholar PubMed PubMed Central

[29] Biswas A, Ramteke P, Pandey H. 2D carbon nano material based gentamicin nano composite for antibacterial activity against Escherichia coli: A novel strategy towards multidrug resistant bacteria. Pharma Innov. 2019 Mar [cited 2021 May 2];5(3.4):397–401. www.thepharmajournal.com.10.22271/tpi.2019.v8.i3.4.08Search in Google Scholar

[30] Wang K, Liu T, Lin R, Liu B, Yang G, Bu X, et al. Preparation and in vitro release of buccal tablets of naringenin-loaded MPEG-PCL nanoparticles. RSC Adv. 2014 Aug [cited 2021 May 2];4(64):33672–9. https://pubs.rsc.org/en/content/articlehtml/2014/ra/c4ra04920a.10.1039/C4RA04920ASearch in Google Scholar

[31] Zaltariov M, Filip D, Varganici C-D, Macocinschi D. ATR-FTIR and thermal behavior studies of new hydrogel formulations based on HPMC/PAA polymeric blends. Cellul Chem Technol. 2018;52(7–8):619–31.Search in Google Scholar

[32] Tan YJ, Lee CS, Er HM, Lim WH, Wong SF. In-vitro evaluation of griseofulvin loaded lipid nanoparticles for topical delivery. J Drug Deliv Sci Technol. 2016 Feb;31:1–10.10.1016/j.jddst.2015.11.002Search in Google Scholar

[33] Gholizadeh H, Messerotti E, Pozzoli M, Cheng S, Traini D, Young P, et al. Application of a thermosensitive in situ gel of chitosan-based nasal spray loaded with tranexamic acid for localised treatment of nasal wounds. AAPS PharmSciTech. 2019 Oct [cited 2022 Jun 27];20(7):299. https://pubmed.ncbi.nlm.nih.gov/31482286/.10.1208/s12249-019-1517-6Search in Google Scholar PubMed

[34] Chin LY, Tan JYP, Choudhury H, Pandey M, Sisinthy SP, Gorain B. Development and optimization of chitosan coated nanoemulgel of telmisartan for intranasal delivery: A comparative study. J Drug Deliv Sci Technol. 2021 Apr [cited 2021 Jan 19];62:102341. https://linkinghub.elsevier.com/retrieve/pii/S1773224721000228.10.1016/j.jddst.2021.102341Search in Google Scholar

[35] Ng SF, Leow HL. Development of biofilm-targeted antimicrobial wound dressing for the treatment of chronic wound infections. Drug Dev Ind Pharm. 2015 Nov;41(11):1902–9.10.3109/03639045.2015.1019888Search in Google Scholar PubMed

[36] Hudzicki J. Kirby-Bauer disk diffusion susceptibility test protocol. Am Soc Microbiol. 2009 [cited 2021 May 30];15:55–63. https://asm.org/Protocols/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Pro.Search in Google Scholar

[37] Qiu Y, Hamilton SK, Temenoff J. Improving mechanical properties of injectable polymers and composites. In: Injectable Biomaterials. USA: Woodhead Publishing; 2011. p. 61–91.10.1533/9780857091376.1.61Search in Google Scholar

[38] Ban E, Park M, Jeong S, Kwon T, Kim EH, Jung K, et al. Poloxamer-based thermoreversible gel for topical delivery of emodin: influence of P407 and P188 on solubility of Emodin and its application in cellular activity screening. Molecules. 2017 Feb [cited 2022 May 30];22(2):246. https:/pmc/articles/PMC6155703/.10.3390/molecules22020246Search in Google Scholar PubMed PubMed Central

[39] Dini V, Salvo P, Janowska A, Francesco FD, Barbini A, Romanelli M. Correlation between wound temperature obtained with an infrared camera and clinical wound bed score in venous leg ulcers. Wounds. 2015 [cited 2022 May 30];27(10):274–8. https://www.hmpgloballearningnetwork.com/site/wounds/article/correlation-between-wound-temperature-obtained-infrared-camera-and-clinical-wound-bed-score.Search in Google Scholar

[40] Kim J-T, Lee D-Y, Kim Y-H, Lee I-K, Song Y-S. Effect of pH on swelling property of hyaluronic acid hydrogels for smart drug delivery systems. J Sens Sci Technol. 2012 Jul [cited 2022 May 30];21(4):256–62. 10.5369/JSST.2012.21.4.256.Search in Google Scholar

[41] Saghazadeh S, Rinoldi C, Schot M, Kashaf SS, Sharifi F, Jalilian E, et al. Drug delivery systems and materials for wound healing applications. Adv Drug Deliv Rev. 2018 Mar;127:138–66.10.1016/j.addr.2018.04.008Search in Google Scholar PubMed PubMed Central

[42] Makwana SB, Patel VA, Parmar SJ. Development and characterization of in-situ gel for ophthalmic formulation containing ciprofloxacin hydrochloride. Results Pharma Sci. 2016 Jan;6:1–6.10.1016/j.rinphs.2015.06.001Search in Google Scholar PubMed PubMed Central

[43] Réeff J, Gaignaux A, Goole J, De Vriese C, Amighi K. New sustained-release intraarticular gel formulations based on monolein for local treatment of arthritic diseases. Drug Dev Ind Pharm. 2013 Nov [cited 2022 May 30];39(11):1731–41. https://pubmed.ncbi.nlm.nih.gov/23078519/.10.3109/03639045.2012.730529Search in Google Scholar PubMed

[44] Nguyen DT, Dinh VT, Dang LH, Nguyen DN, Giang BL, Nguyen CT, et al. Dual interactions of amphiphilic gelatin copolymer and nanocurcumin improving the delivery efficiency of the nanogels. Polymers (Basel). 2019 May [cited 2022 May 30];11(5):814. https://pubmed.ncbi.nlm.nih.gov/31067644/.10.3390/polym11050814Search in Google Scholar PubMed PubMed Central

[45] Yin J, Luo K, Chen X, Khutoryanskiy VV. Miscibility studies of the blends of chitosan with some cellulose ethers. Carbohydr Polym. 2006 Feb;63(2):238–44.10.1016/j.carbpol.2005.08.041Search in Google Scholar

[46] Demeter Á. Thermal analysis of pharmaceuticals. editors. Craig DQ, Reading M. In: ChemMedChem. John Wiley & Sons, Ltd; 2008 [cited 2022 May 30]. Vol. 3. p. 1139–40. https://onlinelibrary.wiley.com/doi/full/10.1002/cmdc.200800161.10.1002/cmdc.200800161Search in Google Scholar

[47] Douzandeh-Mobarrez B, Ansari-Dogaheh M, Eslaminejad T, Kazemipour M, Shakibaie M. Preparation and evaluation of the antibacterial effect of magnetic nanoparticles containing gentamicin: A preliminary in vitro study. Iran J Biotechnol. 2018 [cited 2022 May 30];16(4):287–93.10.21859/ijb.1559Search in Google Scholar PubMed PubMed Central

[48] Unsalan O, Erdogdu Y, Gulluoglu MT. FT-Raman and FT-IR spectral and quantum chemical studies on some flavonoid derivatives: Baicalein and Naringenin. J Raman Spectrosc. 2009 May [cited 2022 May 30];40(5):562–70. https://onlinelibrary.wiley.com/doi/full/10.1002/jrs.2166.10.1002/jrs.2166Search in Google Scholar

[49] de Oliveira SA, da Silva BC, Riegel-Vidotti IC, Urbano A, de Sousa Faria-Tischer PC, Tischer CA. Production and characterization of bacterial cellulose membranes with hyaluronic acid from chicken comb. Int J Biol Macromol. 2017 Apr;97:642–53.10.1016/j.ijbiomac.2017.01.077Search in Google Scholar PubMed

[50] Mahdavinia GR, Ettehadi S, Amini M, Sabzi M. Synthesis and characterization of hydroxypropyl methylcellulose-g-poly(acrylamide)/LAPONITE® RD nanocomposites as novel magnetic- and pH-sensitive carriers for controlled drug release. RSC Adv. 2015 May [cited 2022 May 30];5(55):44516–23. https://pubs.rsc.org/en/content/articlehtml/2015/ra/c5ra03731j.10.1039/C5RA03731JSearch in Google Scholar

[51] Karolewicz B, Górniak A, Owczarek A, Zurawska-Płaksej E, Piwowar, Pluta AJ. Thermal, spectroscopic, and dissolution studies of ketoconazole-Pluronic F127 system. J Therm Anal Calorim. 2014 Mar [cited 2022 May 30];115(3):2487–93. https://link.springer.com/article/10.1007/s10973-014-3661-2.10.1007/s10973-014-3661-2Search in Google Scholar

[52] Pandey M, Choudhury H, Abdul-Aziz A, Bhattamisra SK, Gorain B, Carine T, et al. Promising drug delivery approaches to treat microbial infections in the vagina: a recent update. Polymers (Basel). 2020 Dec [cited 2021 Nov 23];13(1):26. https://www.mdpi.com/2073-4360/13/1/26/htm.10.3390/polym13010026Search in Google Scholar PubMed PubMed Central

[53] Choudhury H, Gorain B, Pandey M, Chatterjee LA, Sengupta P, Das A, et al. Recent update on nanoemulgel as topical drug delivery system. J Pharm Sci. 2017 Jul;106(7):1736–51. http://www.scopus.com/inward/record.url? eid = 2-s2.0-85019135257&partnerID = MN8TOARS10.1016/j.xphs.2017.03.042Search in Google Scholar PubMed

[54] Yourong T, Zhang H, Cao D, Xia L, Du R, Shi Z, et al. Study on cohesion and adhesion of high-viscosity modified asphalt. Int J Transp Sci Technol. 2019 Dec;8(4):394–402.10.1016/j.ijtst.2019.04.001Search in Google Scholar

[55] Salehi M, Ehterami A, Farzamfar S, Vaez A, Ebrahimi-Barough S. Accelerating healing of excisional wound with alginate hydrogel containing naringenin in rat model. Drug Deliv Transl Res. 2021 Feb [cited 2022 May 30];11(1):142–53. https://pubmed.ncbi.nlm.nih.gov/32086788/.10.1007/s13346-020-00731-6Search in Google Scholar PubMed

[56] Hwang MR, Kim JO, Lee JH, Kim Y, IlKim, Chang JH, SW, et al. Gentamicin-loaded wound dressing with polyvinyl alcohol/dextran hydrogel: Gel characterization and in vivo healing evaluation. AAPS PharmSciTech. 2010 Sep [cited 2022 May 30];11(3):1103.10.1208/s12249-010-9474-0Search in Google Scholar PubMed PubMed Central

Received: 2023-03-30

Revised: 2023-06-07

Accepted: 2023-06-21

Published Online: 2023-07-20

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/chem-2022-0357

Keywords for this article

stimuli-responsive hydrogel; gentamicin and naringenin; Box–Behnken statistical design; zone of inhibition; cytotoxicity; application to chronic wound

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