A hydrogel based herbal nanoformulation for enhanced wound healing

Abstract

The aim of the present study is to develop a chitosan based herbal formulation using rhubarb for wound healing. Wound healing is a dynamic process facilitating the regeneration or repair of broken tissue. Rhubarb is commonly used worldwide for their effective antiseptic, antifungal and antiviral properties. Rhubarb loaded chitosan nanoparticles were prepared by ionic cross-linking process with sodium lauryl sulphate. The optimized formulation was examined for antibacterial activity against S. aureus and P. aeruginosa. Wound healing effects of the developed formulation was studied on excision wound model in Wistar albino rats. Average particle size and zeta potential of chitosan nanoparticles varied from 93 to 2,225 nm and from+30.82 to -16.08 mV, respectively. On the basis of particle size, zeta potential, and % entrapment, Formulation C ₄ (0.0595% chitosan, 0.535% cross-linker, and 1% drug) was selected among different checkpoint formulation and further studies were performed. Hydrogel was developed using a combination of polyvinylpyrrolidone and carboxymethyl cellulose. Nanoformulation based hydrogel showed significantly enhanced antimicrobial activity against tested pathogenic microbial strains in comparison to drug solution, as well as marketed formulations.

Keywords:
Nanoparticles; Rhubarb; Design Expert; Wound healing

INTRODUCTION

Wounds are inescapable events of life, which arise due to physical or chemical injury or microbial infections (Ikobi et al., 2012). Wound healing is a dynamic process facilitating the regeneration or repair of broken tissue (Farahpour, Habibi, 2012). Normal wound-healing response begins with injury and is a cascading sequence of events. The healing cascade is activated when platelets aggregate and release of clotting factors consequently resulting in the deposition of fibrin clot at the site of injury (Eppley, Woodall, Higgins, 2004). The fibrin clot serves as a provisional matrix and sets the stage for the subsequent events of healing. Inflammatory cells also arrive along with the platelets at the site of injury providing key signals known as cytokines or growth factors. Fibroblast is the connective tissue responsible for collagen deposition that is needed to repair the tissue injury. In normal tissues, collagen provides strength, integrity, and structure. When tissues are disrupted following injury, collagen is needed to repair the defect and restore anatomic structure and function (Madhava, Sivaji, Tulasirao, 2008; Dos, Soosairaj, 2018).

Effective dressing should have certain characteristics optimized for particular type of wound at a reasonable low cost, with minimum inconvenience to the patient (Ahmed, Ikram, 2016). The Food and Drug Administration approved wound healing dressings based on several polymers such as collagen, silicon, chitosan, and hyaluronic acid (Dreifke et al., 2015). Among various polymers, chitosan exhibits outstanding properties such as non-toxicity, antimicrobial and anti-inflammatory nature besides biocompatibility and biodegradability (Cheung et al., 2015). It accelerates infiltration of polymorphonuclear neutrophil (PMN) cells at the early stage of wound healing, followed by the production of collagen by fibroblasts (Dai et al., 2011). Thus, chitosan is being investigated by several research groups worldwide in the quest of an ideal material for wound healing (Chhabra et al., 2017). A wide variety of topical formulations ranging from solids to semisolids and liquid preparations are available to clinicians and patients. Within the major group of semisolid preparations, the use of hydrogel has expanded, both in cosmetics and in pharmaceutical preparations (Gowda et al., 2016). Hydrogels are appropriate for wound healing due to their ease of administration, wound protection, water retention, and oxygen permeability.

Hydrogel based systems, when applied at pathological sites offer great advantage due to faster release of drug directly to the site of action, with dependence on water solubility of the drug as compared to creams and ointments (Gowda et al., 2016). Hydrogels are hydrophilic, three dimensional networks, held together by chemical or physical bonds. Due to the composition and mechanical aspects, properties of the hydrogel are similar to the natural extracellular matrix, so the hydrogel not only serves as supporting material of cells in the tissue regeneration process, but also deliver a drug payload (Liu et al., 2018).

Chitosan is considered as an ideal material for hydrogels due to its biological adhesiveness and activity, hemostatic effect, as well as its high chemical versatility (Zubair, Malik, Ahmad, 2011). Being positively charged, chitosan can interact with negatively charged molecules of bacterial membrane such as proteins, anionic polysaccharides and nucleic acids and contributes to disruption and lysis of cell (Bhattarai, Gunn, Zhang, 2010). Chitosan-based materials usually exhibit a positive charge (at typical wound pH values), film-forming capacity, mild gelation characteristics and strong wound tissue adhesive property (Jayakumar et al., 2011). Chitosan can interact with mucus and epithelial cells, and finally result in the opening of tight cellular junctions thus increasing the paracellular permeability of the epithelium. In addition, other structural elements of this polymer are likely to contribute to their penetration-enhancing activity (Amidi et al., 2010).

Medicinal plants have been identified and used throughout the human history. The phyto-medicines for wound healing are highly affordable and are generally considered safe as hyper sensitivity reactions are rarely observed with the use of these agents (Patel et al., 2013). The rhubarb plant (Rheum rhabarbarum L.), which belongs to the Polygonaceae family has several reported pharmacological activities such as antitumoral, antimutagenic, anti-inflammatory, and anticarcinogenic activity besides wound healing activity (Jang et al., 2018; Hammond, 2004). The major phytoconstituents reported from the root of rhubarb are anthraquinones (emodin, aloe-emodin, rhein, chrysophenol and physcion), anthrones, flavonoides, lignins, phenols, tannins, saponins, terpenes and trace elements. Rheum emodin has shown significant decrease in all inflammatory markers with increased accumulation of DNA and protein at the wound site (Ahmad et al., 2017).

The aim of the present work is to formulate and evaluate chitosan nanoparticles loaded hydrogel based formulation using novel herbal combination for wound healing.

METHODOLOGY

Material

Chitosan (CS; Mw: 60,000; CAT No: 448869; ≥75% deacetylated), Sodium carboxymethyl cellulose (CMC; Mw: 250,000; CAT No: 419311), Polyvinyl pyrollidone (PVP; Mw: 360,000; CAT No: 81440) and Chloral hydrate (CAT No: C8383) were purchased from Sigma Aldrich. Sodium lauryl sulphate (SLS; CAT No: L5750) and sodium tripolyphosphate (STPP; CAT No: 238503) was obtained from Techno Pharmchem Haryana, India. Tween 80 was procured from LOBA ChemiePvt Limited. Organic solvents were bought from Merck (India). Water used was purified by reverse osmosis (MilliQ, Millipore, USA). All other chemicals were of analytical grade. Rhubarb root was bought from a local market in Taif, Saudi Arabia. Roots (approximately 1 kg) were freeze dried at -60ºC and 0.05 m Bar for 48 h (Telstar, Germany). The dried rhubarb was ground to a coarse brownish powder (50 g) in a blender.

Method

Rhubarb Powder extraction

The stems of Rhubarb (Rheum palmatum) were obtained from local market in Taif, Saudi Arabia. The dried rhubarb stem was ground to coarse powder using a grinder (Philips HL7756/00). Rhubarb powder (50 gm) was extracted with 200 mL ethanol and water (80: 20) solvent by cold extraction process. The extraction was repeated twice and filtered using Whatman filter paper (No. 542, pore size 2.7 μm). The filtrate was dried using rotary evaporator at room temperature and then freeze-dried at -48ºC for 24 h. The dried extract was weighed, and percentage yield was determined by using formula:

$Yield\left(g/100g\right)=\left(W_1\times 100\right)/W_2$

where W ₁ is the weight of the extract residue obtained after solvent solution (1 mL) was added drop wise with a syringe into the chitosan solution under magnetic stirring at 800 rpm, at room temperature, for 10 minutes. To prepare drug loaded nanoparticles, dried rhubarb extract (100 mg) was dissolved in 1 mL ethanol. Thereafter the drug solution consisting of rhubarb was mixed with 1 mL cross-linker solution. Drug and cross-linker solution were added drop-wise into 10 mL each of varied concentration chitosan solution (0.05%w/v, 0.075%w/v and 0.1%w/v) consisting of 0.5% v/v tween 80. The cross-linking process was accomplished using the same procedure as used to prepare the placebo nanoparticles.

Experimental Design

Box-Behnken design was used to optimize the formulation parameters and systematically investigate the wide range effects of independent and dependent variables. Polymer concentration (X ₁), cross-linker concentration (X ₂) and drug concentration (X ₃) were three independent variables (factors) considered in the preparation of chitosan nanoparticles, while the average particle size (Y ₁), zeta potential (Y ₂), drug entrapment (Y ₃) and drug release (Y ₄) were dependent variables.

Preparation of chitosan nanoparticles

The chitosan nanoparticles were prepared through the procedure described by Agarwal et al., 2015. The CS nanoparticles were obtained by inducing gelation of a CS solution with STPP. Ionotropic gelation takes place due to the interaction between positively charged amino groups and negatively charged STPP. Chitosan solution (10 mL) was prepared by dissolving weighed amount of chitosan (50 mg, 75 mg, 100 mg)in 1% acetic acid solution (10 mL) followed by mild stirring and heating at about 60ºC overnight to form clear chitosan solution (0.05%w/v, 0.075%w/v and 0.1%w/v). Tween 80 [0.5% (v/v)] was added to chitosan solutions as a stabilizer. Sodium tripolyphosphate solution/Sodium lauryl sulphate (0.25%w/v) was prepared by dissolving weighed amount of cross-linker in 10 mL of deionized water. Cross-linking (response). For each factor, the experimental range based on the result of preliminary experiment was selected and process parameters were studied by conducting the runs at different level of all factors. Data collected for responses in each run were analyzed using the software Design Expert 11 (Statease, USA) and designed into a multiple linear regression model.

Evaluation of chitosan nanoparticles

Particle size and zeta potential

Particle size distribution and zeta potential of chitosan nanoparticles were measure by Zetasizer Nano S (Malvern, UK). The analysis was carried out at 90º scattering angle and a temperature of 25ºC, using nanoparticles dispersed in de-ionized distilled water (2 mL of chitosan nanoparticles was dispersed in 5 mL of deionized water and bath sonicated) (Asasutjarit et al., 2013).

Percent entrapment efficiency

Percent entrapment efficiency of rhubarb was calculated using method described by Asasutjarit and coworkers in 2013 by using the following equation:

$\%EE=\left\{totaldrugcontent-freedrugcontent/totaldrugcontent\right\}\times 100$

Briefly, a suspension of a representative drug loaded nanoparticles was centrifuged at 12,000 rpm for 2 hours. The supernatant was collected for determination of Rhubarb content. The drug was analyzed spectrophotometrically at λmax of 314 nm (Shimadzu 1800).

Percent drug release

In vitro release study of Rhubarb from chitosan nanoparticles was carried out in phosphate buffer medium (pH: 6.8) for 6 hours. 2 mL of each formulation was filled in the dialysis bag (previously activated by overnight submersion in the release medium) and inundated into 100 mL phosphate buffer solution with constant stirring at 100 rpm. At a predetermined time intervals of 0, 0.5, 1, 2, 3, 4, 5 and 6 h, 1 mL of buffer solution was withdrawn using a syringe fitted with 0.22 μm filter and replaced with fresh buffer solution to maintain sink condition. The drug release was assayed by UV-visible spectrophotometer at λmax value of 314 nm. The experiments were carried out in triplicate and average values were taken (Wani, Raza, Khan, 2013; Sun et al., 2015).

Freeze drying

Ten milliliters of placebo and drug loaded chitosan nanosuspension were freeze dried using freeze drier (Heto Drywinner, Denmark) at -55ºC temperature under vacuum (5 m Torr).

Scanning electron microscopy (SEM)

The chitosan nanoparticles were examined by SEM for determining the surface morphology. The placebo and drug loaded chitosan nanoparticles were freeze dried and gold sputter coating was carried out under reduced pressure in an inert argon gas atmosphere (Agar Sputter Coater P7340). After coating, the sample was examined under scanning electron microscope (Leo 435 VP) operated at 15-25 KV and the photographs were documented.

Transmission electron microscopy (TEM)

To prepare sample for TEM analysis, placebo and drug loaded chitosan nanoparticles were dispersed in distilled water and ultrasonicated for a minute to de-aggregate the particles. A small drop of a suspension was then placed on carbon coated grid covered with nitrocellulose, stained with phosphotungstic acid (PTA) and dried at room temperature. Nanoparticle analysis was carried out by Hitachi H 9000 NAR transmission electron microscope with resolution of 0.18 nm at 300 kV in the phase contrast HRTEM imaging mode equipped with an energy dispersive X-ray (EDX).

Preparation of hydrogel

The hydrogels were prepared by moist heat treatment using aqueous solution of PVP and CMC in varying ratios (0: 100, 20: 80, 50: 50, 80: 20, and 100: 0). The hydrogel solutions (50 mL) were prepared in 250 mL sealed glass bottles under physical stimulations in an autoclave (15 lbs pressure and 120ºC temperature for 20 min). Prepared hydrogels were evaluated for swelling studies in order to get the final formulation.

Evaluation of hydrogel

Equilibrium Swelling Study

The 5-g gel was filled in emptied and previously weighed tea bag. The experiment was carried out by measuring the weight gain as a function of immersion time in 100 mL of buffer solution, pH 6.8. Measurements were made until equilibrium hydration degree was reached, when three consecutive determinations gave the same weight. Before the final weight measurement, the tea bag was hung up to 15 min in order to remove the excess immersion fluid. The equilibrium swelling was calculated by dividing the difference in weight of swollen gel to that of dried gel by weight of dried gel.

$Degreeofswelling\left(\%\right)=M_F-M_I/M_I\times 100$

M_F=Weight of swollen gel sample

M_I=Initial mass of sample

Antimicrobial activity

Microbial strains

Pure cultures of pathogenic bacterial species used in this study were Staphylococcus aureus, which is a gram-positive bacterium; and Pseudomonas aeruginosa, which is a gram-negative bacterium.

Methods of preparation of test organisms

The test organisms were maintained on slants of nutrient agar medium and transferred to a fresh slant once in a month and were incubated at 37ºC for bacterial culture. Using 10 mL of sterilized normal saline solution, the cells/mycelium were washed from the slants. A dilution factor was determined which gave optical density of 1.5 at 600 nm. The amount of suspension to be added to each 100 mL agar or nutrient broth was determined by use of test plates or test broth. The test organisms were stored under refrigeration.

Anti-microbial assay

In vitro antimicrobial assay was carried out by Agar well diffusion (Namasivayam, Samydurai, Chettia, 2017). A Previously liquefied and sterilized nutrient agar (20 ml) was poured in to petri-plates of 100 mm size (to make uniform thickness) and kept for solidifying. Microbial suspensions were spread over the solidified media. Holes were made in each plate with a stainless steel borer having 6 mm ID.100 μL of rhubarb (0.5% w/w) loaded nanoformulations gel and solution (0.5% w/w rhubarb) was poured into holes while 1% povidone iodine ointment was used as standard. For diluted samples, ten grams of each sample (control, drug solutions and nanoformulations) were transferred to a flask containing 10 mL of sterile saline solution, and homogenized for 2 minutes using a vortex mixer. The wells were filled to about three-quarters full with diluted and undiluted samples and the plates were then left for standing for 3 hours at 4^OC for proper diffusion of the drugs/test solutions. After diffusion process all the petri plates were incubated for 24 h at 37ºC for bacterial culture. After 24 h the plates were examined and the diameter of zones of inhibition was accurately measured.

Evaluation of Wound Healing Activity in rats

The study was approved by Taif University ethical committee (Application No: 40-35-0152) and were carried out in accordance with the ethical guidelines prescribed by U.K. Animals (Scientific Procedures) Act, 1986 (https://www.gov.uk/guidance/guidance-on-the-operation-of-the-animals-scientific-procedures-act-1986). Wound healing activity was carried out according to the procedure described previously (Jagtap et al., 2019).

Animals

Wistar rats (150-200g) of either sex were used for study. All animals had free access to pelleted food and water ad libitum. Temperature was maintained at 24±1^oC.

Treatment

Animals were wounded under light ether anesthesia by intra-peritoneal injection using ketamine anesthesia (30 mg/kg) semi aseptically. As shown in Table I, the animals were assigned into four group (n=6). Group I was untreated group and taken as control. Group II animals received povidone iodine ointment treatment and group III animals received drug solution (0.5% w/w rhubarb dissolved in ethanol). While Group IV was given nanoparticles loaded gel formulation (0.5% w/w rhubarb). No other topical or systemic therapy was given to animals during the course of this study.

TABLE I
Treatment schedule for wound healing activity

Groups	Treatment schedule*
Group-I	Control
Group-II	Povidone-Iodine ointment
Group-III	Rhubarb solution (0.5%w/w)
Group-IV	Chitosan nanoparticles based hydrogel (0.5% w/w)

• *6 animals/group

Excision wound model

Hair were removed from dorsal thoracic central region of anaesthetized rats. Full thickness from the remarketed area was excised to produce wound measuring around 100 mm². Wound was cleaned with cotton swab soaked in alcohol and rats were left undressed to the open environment. Formulations (Povidone iodine ointment, herbal drug solution and herbal nanoformulation) were applied on wounds of different groups (group II, group III and group IV) once daily for 10 days starting from the first day of wounding. The wounds were traced on transparent tracing paper by permanent marker on the day of wounding and subsequently on alternate days until healing were complete. Wound contraction was measured for 10 days at interval of 2 days (Shinde, Ahmed, Singh, 2013).

Statistical Analysis

The data from individual experiments was presented as Mean±S.D. Differences between groups was analyzed using analysis of variance (ANOVA) followed by Dennett’s multiple comparisons test and minimum criterion for statistical significance was set at p < 0.05 for all comparisons. The tests were performed by using GraphPad Prism version 6.01 for Windows, GraphPad Software (Sharma et al., 2014).

RESULTS

Preparation of extract

The cold extraction process was performed to prepare Rhubarb extract and % yield of dried extract was found to be 18.4%.

Preparation of chitosan nanoparticles

Preliminary trials

Stirring method

Placebo chitosan nanoparticles were prepared by ionotropic gelation with STPP using different concentration of chitosan ranging from 0.05% to 0.5% w/v. Results for an average particle size showed all particles were in micrometer range. 0.5% chitosan concentration yield large clumps or aggregated particles.

Probe Sonicator

Placebo chitosan nanoparticles were prepared using probe sonicator and results are shown in Table II. It was observed that chitosan concentration when used in the range of 0.025% to 0.1% produced small size nanoparticles. While when polymer concentration increased to 0.5%, it produced large aggregates which was not detected by Malvern zetasizer.

TABLE II
Average particle size of placebo chitosan nanoparticles* using probe sonicator

S. No	Chitosan concentration	Average particle size
1	0.025%	158±2.5 nm
1	0.05%	162±3.7 nm
2	0.1%	368±4.5 nm
3	0.5%	Aggregates**

• *All formulations were prepared at 0.25% TPP
• **When particles could not be detected by Malvern zetasizer, it has been considered as aggregates

Drug loaded Chitosan nanoparticles using TPP as crosslinker: When drug loaded formulations prepared using TPP as crosslinker, all particles were in µm size range. Therefore it was decided to use SLS (sodium lauryl sulphate) instead of TPP.

Drug loaded formulation using SLS as a crosslinker: Drug loaded nanoparticles were prepared using SLS as a crosskinker and results are shown in Table III. This time the formulation were nanosized. Therefore SLS was selected as a cosslinker and polymer concentration from 0.025% w/v to 0.1% w/v was selected for further optimization by Design Expert. Drug concentration was selected from 0.1% w/v (10 mg) to 1% (100 mg) with respect of chitosan solution which was 10 mL (for both drugs) according to prior art.

TABLE III
Average particle size of drug loaded chitosan nanoparticles*using SLS as a cross-linker

S. No	Chitosan concentration	Average particle size
1	0.025%	86±2.33 nm
1	0.05%	223±2.14 nm
2	0.1%	563±4.5 nm
3	0.5%	Aggregates**

• *All formulations were prepared at 0.25% SLS and 0.5%w/v (with respect of chitosan volume) of rhubarb
• **When particles could not be detected by Malvern zetasizer, it has been considered as aggregates

Experimental Design

Box-Behnken designs are popular designs for use in response-surface exploration where Response Surface Methodology (RSM) was used to systemically investigate the effect of a wide range of independent and dependent variables. The details of design are shown in Table IV. Polymer concentration, cross-linker concentration and drug concentration were 3 independent variables considered in the preparation of nanoparticles while particle size, zeta potential, drug entrapment and drug release were dependent variable. To identify the optimum levels of different process parameters influencing the particle size, an experimental design of 17 runs containing central points was made according to the Box-Behnken design for these selected parameters. The individual and interactive effects of different process variables were studied by conducting the process at different levels of all factors. All the responses observed in 17 runs were simultaneously fitted to first order-, second order- and quadratic models using DESIGN EXPERT. It was observed that the best-fitted model was quadratic model. Analysis of variance for the response is represented in Table V.

TABLE IV
Level of process parameters used in experiment

Code	Process Parameters	Levels
		-1	0	+1
X 1	Polymer concentration (%)	0.05	0.075	0.1
X 2	Cross-linker concentration (%)	0.25	0.625	1.0
X 3	Drug concentration (%)	0.25	0.625	1.0

TABLE V
Analysis of variance of calculated model for response (Y ₁: Particle size)

Results of the analysis of variance	Particle size	Zeta potential	Percent entrapment	Percent drug release
Regression
Sum of squares	0.00075	3375.15	723.68	3928.45
df	9	9	9	9
Mean Squares	0.000083	375.02	80.41	436.49
F-ratio	2235.24	969.46	155.36	128.57
P	<0.0001	<0.0001	<0.0001	<0.0001
Residual
Sum of squares	2597.95	2.71	3.62	23.77
df	7	7	7	7
Mean Squares	371.14	0.3868	0.5136	3.40
SD	19.26		0.7194	1.84
Lack of Fit test	Non significant	Non significant	Non significant	Non significant
Sum of squares	2120.75	1.80	1.24	15.89
df	3	3	3	3
Mean Squares	706.92	0.5986	0.4132	5.30
F value	5.93	2.63	0.6936	2.69
Correlation of coeffecient (R2)	0.9997	0.9992	0.9950	0.9940
Correlation of varaition (CV %)	2.63	3.98	1.32	2.69

• Analysis of variance from DESIGN EXPERT 7.0.
• Df: Degree of freedom.

The Model F-valuesfor different responses as shown in Table V implies the model is significant. The Lack of Fit F-values for all responsesimplies that Lack of Fit is not significant. A correlation plot between actual and predicted values for different responses are shown in Figure 1. The Predicted R² valueis in reasonable agreement with the Adjusted R²; i.e. the difference is less than 0.2. Adeq Precision measures the signal to noise ratio. A ratio greater than 4 is desirable and thus indicate an adequate signal. This model can be used to navigate the design space.

FIGURE 1
A correlation plot between actual and predicted values where a: particle size, b: zeta potential, c: percent entrapment, d: percent drug release.

Evaluation of chitosan nanoparticles

Average particle size and zeta potential

A positive value of X ₁ (Equation 1) represents a favorable optimization process while negative value of X ₂ indicate an inverse relationship. The results showed that an increase in polymer concentration resulted in an increase in response, i.e, average particle size. Formulation C ₃ prepared with 0.05% concentration showed an average particle size of 225 nm when compared to formulation C ₄ which showed a marked increase in average particle size (1,887 nm) at a constant volume of cross-linker and drug concentration. The expected fact might be due to increase in viscosity of solution with an increase in polymer concentration which leads to increase in droplet size upon dispersion and large sized particles upon cross-linking. These results are in agreement with previous literatures which also observed an increase in size of PLGA nanoparticles with increase in polymer concentration (Sharma et al., 2014).

An inverse relationship between crosslinker concentration and particle size was observed. As SLS concentration increases from 0.25% to 1%, particle size also decreases from 636 to 342 nm (C ₉ and C ₁₀). The result can be attributed to the fact that increase in cross-linker concentration increases the crosslinking and shrinking of polymeric particles which will lead to decrease in particle size. Similar results were observed by previous researchers who observed decrease size of protein nanoparticles with an increase in crosslinker due to formation of denser nanoparticles (Nahar et al., 2008; Yien et al., 2012). A significant positive value of X ₃ indicates that the drug concentration had a marked influence on particle size. An increase in drug concentration resulted in an increase in viscosity of the solution, which resulted in an increase in droplet size and hence further increase the particle size (Figure 2.a.1). Formulation C₆ prepared with 0.1% drug concentration showed an average particle size of 1527 nm when compared with formulation C₈ (1% drug concentration) which showed an average size of 1954 nm. As clearly depicted all factors were exhibited nearly linear relationship at all levels (Figure 2.b.1).

FIGURE 2
(a) Contour plots (b) Response surface plot showing the effect of different process parameters on (1) average particle size (2) Zeta potential (3) Percent entrapment (4) Percent drug release.

Chitosan concentration showed positive relationship while crosslinker and drug concentration established a negative response for zeta potential values (Equation 2). As chitosan concentration increased from 0.05% to 0.1%, zeta potential values increased from -17.57 mV (Formulation C ₃) to+13.35 mV (Formulation C ₄). Zeta potential of chitosan nanoparticles showed a net positive surface charge due to excess positive charge (amino gropus) of chitosan (Baker, Grant, 2018). As crosslinker concentration increased from 0.25% to 1%, zeta potential decreases from 32.5 mV (Formulation C ₂) to 13.35 mV (Formulation C ₄) due to interaction with negative charge SLS. Similar negative relationship was observed with drug concentration due to formation of negative charge oxygen atom after dissociation of phenolic glycosides of rhubarb. This negative behavior can further be depicted by the zeta potential value of diluted 0.1% drug solution alone which was found to be -44. 2±3.05 mV. Such inverse relation was established by contour plot (Figure 2.a.2). A similar negative interactive relationship is shown in RSM plot (Figure 2.b.2).

Percent drug entrapment

A significant positive value of X ₁ indicates that the polymer concentration had a marked influence on percent drug entrapment (Equation 3). As polymer concentration increased from 0.05% w/v to 0.1% w/v, drug entrapment also increased from 53.86% (Formulation C₇) to 68.23% (Formulation C₈), respectively. The reasons can be explained on the basis of viscosity of solution with increase in polymer concentration. Firstly, high viscous nanoparticles shell will be formed at higher concentration which prevent loss of drug during cross-linking process. Secondly with increase in vicosity, an increase in particle size was observed which leads to high entrapment. Thus high entrapment with formulation C₈ can be explained on the basis of large particle size which was 1954 nm when compared with formulation C₇, where average particle size is 98 nm. Similarly, we observed inverse relationship with crosslinker and positive relationship with drug concentration. Formulation C₉ prepared with 0.25% cross-linker resulted in high entrapment ie 51.45% when compared with formulation C₁₀ where entrapment was significantly low ie 36.76%. Increase in cross-linker concentration decreases the entrapment due to shrinking process taken place during cross-linking reaction which resulted in loss of drug. The second possible explaination will be again supported by the particle size. Formulation C₉ showed an increase particle size ie 636 nm and thus high entrapment when compared with formulation C₁₀ where particle size is significantly low ie 342 nm. Increase in drug concentration had a positive influence on response. This again can be explained on the basis of viscosity of solution and particle size. An increase in drug concentration resulted in an increase in particle size and hence an increase in drug entrapment. Formulation polymer concentration which slowed the diffusion of drug molecules. Another explanation for the hypothesis was increase particle size with increase in polymer concentration which extends the diffusion time for molecules due to increase in diffusion pathlength. Increase in cross-linker concentration decreases the percent drug release due to formation of more strenthen nanoparticles. Formulation C ₆ prepared with 0.1% drug concentration showed 56.08% drug release when compared with formulation C ₈ prepared with 1% drug concentration which showed only 46.24% drug release in the same time. The reason may be an increase in particle size with an increase in drug concentration and thus decrease in the percent drug release. But this reason alone can not support the hypothesis as increment in particle size only slighly ie from 1,527 nm (Formulation C ₆) to 1,954 nm (Formulation C ₈). This interative effect of chitosan and crosslinker on percent drug release can be clearly depicted by contour plots in Figure 2.a.4. Similar interactive inverse relationship at all levels can be seen clearly in RSM plot (Figure 2.b.4).

Validation of design results

Point prediction of the design expert software was C₁₀ showed small particle size (342 nm) and less drug used to determine the optimum values of the factors for entrapment (36.76%) when compared with formulation different responses. Table VII lists the composition of the C₁₂ (average particle size: 693 nm), where comparatively checkpoints, their predicted and experimental values of the high entrapment (55.68%) was observed.Similarly contour and RSM plot showed almost linear relationship between the interactive factors at all levels (Figure 2.a.3 and Figure 2.b.3).

TABLE VI
Box-Behnken Statistical design with results

Code	Process Parameters			Average Particle Size (nm)	Zeta potential(mV)	Entrapment (%)	Drug release (T6h)
	X1	X2	X3
C 1	0.05	0.25	0.55	331	27.23	51.22	97.7
C 2	0.1	0.25	0.55	2118	32.5	56.71	53.19
C 3	0.05	1	0.55	225	-17.57	45.22	67
C 4	0.1	1	0.55	1887	13.35	56.52	42.38
C 5	0.05	0.625	0.1	63	10.13	52.38	88.42
C 6	0.1	0.625	0.1	1527	27.88	53.21	56.08
C 7	0.05	0.625	1	98	8.7	53.86	77.49
C 8	0.1	0.625	1	1954	16.3	68.23	46.24
C 9	0.075	0.25	0.1	636	31.95	51.45	99.16
C 10	0.075	1	0.1	342	-12.95	36.76	68.23
C 11	0.075	0.25	1	753	13.19	49.79	75.14
C 12	0.075	1	1	693	-6.46	55.68	62
C 13	0.075	0.625	0.55	362	20.84	58.11	66.18
C 14	0.075	0.625	0.55	383	20.82	58.45	67.45
C 15	0.075	0.625	0.55	377	20.6	58.77	65.42
C 16	0.075	0.625	0.55	369	20.82	57.36	65.19
C 17	0.075	0.625	0.55	356	20.8	59.44	68.50

• The results of experimental data and simulated values for all responses are listed in Table VI. The model proposes the following polynomial equations (only significant terms are included) for different responses: Y ₁(average particle size)=+369.40+846.12X ₁-86.37X ₂+116.25X ₃-31.25X ₁ X ₂+98X ₁ X ₃+58X ₂ X ₃+537.68X ₁2+233.18X ₂2(1) Y ₂(Zeta potential)=+20.78+6.38X ₁-16.06X ₂-3.16X ₃+9.05X ₁ X ₂-2.54X ₁ X ₃-6.31X ₂ X ₃+1.21X ₁2-8.11X ₂2-6.23X ₃2 (2) Y ₃(percent entrapment)=+58.43+4X ₁-1.87X ₂+4.22X ₃+1.45X ₁ X ₂+3.39X ₁ X ₃+5.15X ₂ X ₃+1.45X ₁2-7.25X ₂2-2.75X ₃2(3) Y ₄(percent drug release)=+66.55-16.59X ₁-10.70X ₂-6.38X ₃+4.97X ₁ X ₂+0.2725X ₁ X ₃+4.45X ₂ X ₃-5.28X ₁2+3.80X ₂2+5.79X ₃2(4)

TABLE VII
Optimized parameters with predicted and experimental values for different response and percentage prediction error

	Parameters	Predicted values	Experiment Values	Experimental validity*	Prediction error (%)**
Formulation CP 1	Particle size	279.47 nm	279.02 nm	99.83	+0.161
	Zeta potential	20.99 mv	20.2 mV	96.23	+3.76
	% Entrapment	58.3%	56.23 %	96.44	+3.55
	% Drug release	70.56%	68.13%	96.55	+1.47
Formulation CP 2	Particle size	337.8 nm	367.1 nm	108.67	-8.67
	Zeta potential	12.81 mv	13.3 mV	103.82	-3.82
	% Entrapment	57.46%	57.21 %	99.56	+0.43
	% Drug release	70.78%	67.42%	95.25	+4.75
Formulation CP 3	Particle size	214.95nm	208 nm	96.76	+3.23
	Zeta potential	20.79 mV	18.5 mV	89.94	+11.01
	% Entrapment	56.09%	55.20%	98.41	+1.59
	% Drug release	69.03%	68.14%	98.71	+1.28
Formulation CP 4	Particle size	125.87nm	107 nm	85.0	+14.99
	Zeta potential	12.69 mv	11.6 mV	91.41	+8.59
	% Entrapment	54.81%	54.11%	98.72	+1.27
	% Drug release	76.50%	74.23%	97.03	+2.96

• CP₁: chitosan 0.07 %, cross-linker concentration: 0.5425% and drug concentration: 0.7%
• CP₂: Chitosan 0.07%, cross-linker 0.54 and drug concentration 1%.
• CP₃: 0.0645% chitosan, 0.4600% cross-linker and drug 0.8020%
• CP₄: 0.0595% chitosan, 0.5350% cross-linker and drug 1%
• *Experimental validity: Experimental values/Predicted values*100
• **% Prediction error: Predicted value-Experimental value/Predicted value*100

Percent drug release

An inverse relationship was observed with all independent variables (chitosan concentration, cross-linker concentration, drug concentration) and reponse (% drug release) (Equation 4). As chitosan concentration increased from 0.05% to 0.1%, percent drug release was decreased from 97.7% (Formulation C ₁) to 53.10% (Formulation C ₂). The most obvious explanation behind this fact was formation of dense matrix with increase in response variables and the percentage error in prognosis. Our point of selection criteria for independent variables includes use of minimum cross-linker and maximum drug concentration. Formulations were optimized on the basis of minimum particle size (<400 nm), positive zeta potential values that favored interaction with negative skin surfaces (>10 mV) and entrapment (>50%). Finally, the optimum values of polymer concentration (0.0595%-0.07 %), cross-linker concentration (0.460% -0.5425%) and drug concentration (0.7%-1%) were obtained. These values predict different values for responses. These predicted values of responses were validated by preparing the nanoparticles with previously optimized process parameters and conducting further in-vitro studies. The experimental values show 85.0% to 108.67% experimental validity for different responses with-8.67% to+14.99% prediction errors. Thus, the low magnitudes of error and the significant values of R₂ (0.745-0.995) in the current study indicated a high prognostic ability of implemented design.

Particle size distribution and zeta potential for different check point formulations are given in Figure 3.

FIGURE 3
(a) Particle size distribution (b) Zeta potential of various checkpoint formulations.

On the basis of particle size and drug concentration, Formulation C ₄ was selected among different checkpoint formulation and proceed for further studies.

Transmission electron microscopy

TEM image of placebo chitosan nanoparticles showed well scattered nano-sized particles (Figure 4.a). Drug loaded chitosan nanoparticles as shown in Figure 4.b clearly demonstrate scattered as well as cluster of nanosized particles.

FIGURE 4
Transmission electron microscopic images of optimized (a) Placebo chitosan nanoparticles (b) drug loaded chitosan nanoparticles (c) 10 times diluted drug loaded chitosan nanoparticles based hydrogel.

Scanning electron microscopy

SEM image of Placebo chitosan nanoparticles showed small size nanoparticles entrapped in chitosan matrix (Figure 5.a). Whereas drug loaded chitosan nanoparticles photomicrographs showed clusters of spherical shaped particles (Figure 5.b). However, some extent of particle clumping and aggregation can be observed which incurred during freeze drying process.

FIGURE 5
Scanning electron microscopic images of optimized (a) Placebo chitosan nanoparticles (b) drug loaded chitosan nanoparticles.

Formulation of hydrogel

The hydrogel were prepared using different PVP and CMC combinations and optimized on the basis of swelling index (Table VIII).

TABLE VIII
Formulae for hydrogel base using different concentration of PVP and CMC

Formulation code	CMC (g)	PVP (g)	Ratio (CMC: PVP)	Consistency
HC 1	2	0	1:0	Gel
HC 2	1.6	0.4	0.8:0.2	Gel
HC 3	1.0	1.0	0.5:0.5	Gel
HC 4	0.4	1.6	0.2:0.5	Thin
HC 5	0	2	0:1	Thin

Formulation HC ₄ and HC ₅ produced gel of liquid consistency and therefore were not proceeded further. Remaining formulations were evaluated for swelling studies.

Evaluation of gel

Swelling studies

Swelling studies is shown in Figure 6. Formulation HC ₁ showed 1.89% swelling in 20 minutes while formulation HC ₃ showed 0% swelling at various time intervals. Formulation HC ₂ prepared with 20% PVP and 80% CMC showed best degree of swelling (7.5% in 40 minutes) and hence selected for further studies.

FIGURE 6
Swelling studies of different hydrogels prepared by combination of PVP and CMC.

Hydrogel loaded chitosan nanoparticles

Formulation CP ₄ for nanoparticles: 0.0595% chitosan, 0.535% cross-linker (SLS) and drug concentration 1% (100 mg rhubarb) Formulation HC ₂ for gel (50 mL): 4gm CMC (80%) and 1gm PVP (20%) 10.56 gm of gel (Formulation HC ₂) was blended with 13 mL of chitosan nanoparticles (Formulation CP ₄) (weighed 9.440 g) to prepare 0.5% w/w of rhubarb loaded gel formulation. The formulation was then evaluated for wound healing activity. A schematic representation for preparation of optimized formulation is shown in Figure 7. TEM photomicrograph of 10 times diluted hydrogel loaded chitosan nanoparticles as shown in Figure 4.c revealed spherical and nanosized particles which still confirmed nanoscopic behavior of the optimized formulation.

FIGURE 7
Schematic representation showing stepwise preparation of hydrogel containing rhubarb loaded chitosan nanoparticles.

Antimicrobial activity

The optimized formulation of rhubarb was examined for antibacterial activity against S. aureus and P. aeruginosa. As shown in Figure 8, nanoformulation showed significant antimicrobial activity against tested pathogenic microbial strains in comparison to solution as well as marketed formulations. Zone of inhibition for nanoformulations was found to be 18.0±0.50 mm which is significantly higher (p<0.0001) in comparison to solution and marketed formulation (povidone iodine ointment) where the reported values were 16.0±0.39 mm and 10.0±0.33 mm, respectively for S. aureus. However, zone of inhibition against P. aeruginosa for solution and nanoformulation was found to be, 12.0±0.35 mm and 14.0±0.38 mm, respectively resulting in statistical differences (p<0.0001) when compared to marketed formulation (9.0±0.19 mm).

FIGURE 8
Comparison of total Inhibition profile (mm) for different antimicrobial formulation against Staphylococcus aureus and Pseudomonas aeruginosa.

Wound healing activity

A schematic diagram for wound healing activity in rats is shown in Figure 9.a. Excision wound model showed that gel formulation exhibited a significant increase in the percentage of wound contraction as compared to control groups over the period of 10 days (p<0.05) (Table IX). Application of hydrogel based chitosan nanoparticles formulation led to 54.63% (p=0.0004) contraction compared with drugs solutions (50.17%, p=0.0024) and standard (22.16%, p=0.1479), whereas control showed 24.34% contraction after 4^th day of treatment (Figure 9.b). After 6^th day, gel formulation led to complete healing of wounds (p=0.0001) when compared with control where only 39.92% wound healing was observed. Whereas standard treatment and drug solutions showed 36.14% (p=0.0724) and 68.88% (p=0.0007) wound healing, respectively. After 8^th day, solution showed 91.71% (p=0.0001) wound healing when compared with standard and control where healing was 79% and 59.62%, respectively. Photographs are presented in Figure 9.c which showing wound contraction through 2-10 days.

FIGURE 9
(a) Schematic representation for wound healing activity in rats (b) Percent wound contraction of different formulations on various days in rats (c) Photographic representation of contraction rate showing percent wound contraction area on different post-excision days of control, marketed formulation, drug solutions and gel formulation.

TABLE IX
Evaluation of wound contraction for excision wounds in rats

Days	Group-I Control (mm2)	Group-II (mm2)	Group-III (mm2)	Group-IV (mm2)
0	102.01±1.42	105.63±1.78	120.70±1.76	113.04±1.11
2	98.22±1.54	97.21±2.33	110.10±2.19	95.18±2.52
p value		0.566	0.0015	0.1492
4	77.18±2.51	82.22±4.18	60.27±3.45	51.28±3.29
p value		0.1479	0.0024	0.0004
6	61.29±3.58	67.45±2.56	37.55±2.55	00
p value		0.0724	0.0007	0.0001
8	41.19±2.63	22.14±3.17	10±1.21	00
p value		0.0013	0.0001	0.0001
10	22.12±4.11	00	0	00
p value		0.0007	0.0007	0.0007

• Values are mean±SD of 3 rats in each group. Group I: control, Group II: marketed formulation, Group III: drug solution and Group IV: gel formulation. P<0.05 considered as statistically significant. All groups compared to respective day control group (statistical analysis was done by one-way analysis of variance)

DISCUSSION

The present study was carried out to prepare novel nano-based formulation using rhubarb and investigate its wound healing properties. Wound healing is the dynamic process which takes place by regeneration or repair of broken tissue. Rhubarb is commonly used worldwide for remarkable antiseptic, antifungal and antiviral properties. Chitosan nanoparticles were initially prepared using ionotropic gelation by STPP under magnetic stirrer. This method produced particles of micron size and was not pursued further. Further attempts to prepare nanoparticles by probe sonicator were successful as the particles produced were in nanometer size range. When loaded with rhubarb the method failed to produce nanometer size formulation. Different concentrations of TPP and chitosan were tried, but only aggregates were obtained. Finally, sodium lauryl sulphate was used as the stabilizer in place of TPP. Sodium lauryl sulfate (SLS) is an anionic surfactant commonly used in detergents and cleaning products. SLS has some antimicrobial activity but is more often a synergist used with other antimicrobial active ingredients. It is used in almost every commercial soap, shampoo, and face wash as a detergent and foaming agent. It is considered moderately toxic, with a probable oral lethal dose for humans estimated to be 500-5,000 mg/kg, between 1 oz to 1pint (or 1 lb) for 70 kg (150 lb) person (Baker, Grant, 2018). So, our aim was to use SLS in minimum concentrations. It has been reported that the addition of 5% SLS to gel formulation containing 3% foscarnet markedly reduced the mean lesion score (Piret et al., 2000).

Box Behnken statistical Design was used to systemically investigate the effect of a wide range of independent and dependent variables, viz; polymer concentration, SLS concentration and drug concentration. To identify the optimum levels of different process parameters influencing the particle size, an experimental design of 17 runs containing central points was made according to the design for these selected parameters. It was observed that the best-fitted model was quadratic model. Finally, using point prediction tool of design expert, the optimum values of polymer concentration (0.0595%-0.07 %), cross-linker concentration (0.460%-0.5425%) and drug concentration (0.7%-1%) were obtained. These predicted values of responses were validated by preparing the nanoparticles with previously optimized process parameters and conducting further in-vitro studies. The experimental values show 85.0% to 108.67% experimental validity for different responses with -8.67% to+14.99% prediction errors and thus indicated a high prognostic ability of the implemented design. On the basis of particle size, zeta potential and % entrapment, Formulation C ₄ was selected among different checkpoint formulations and processed for further studies. SEM and TEM photomicrographs showed well spherical and small sized particle which retained the nanoscopic size when loaded in a hydrogel.

The hydrogel was prepared using different PVP and CMC combinations and optimized on the basis of swelling index. Formulation HC ₂ prepared with 20% PVP and 80% CMC showed best degree of swelling (7.5% in 40 minutes) and hence selected for further studies. Final formulation was prepared by blending 10.56 gm of gel (Formulation HC ₂) with 13 mL of chitosan nanoparticles(Formulation CP ₄) (weighed 9.440 g) to provide 0.5% w/w of rhubarb loaded gel formulation. The formulation was then evaluated for wound healing activity.

The antibacterial activities of Rheum emodi against the bacterial strains Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) were tested. Staphylococcus aureus, a cutaneous pathogen, is responsible for the majority of bacterial skin infections in humans and Pseudomonas aeruginosa is responsible for causing urinary tract infection, gastrointestinal infection, septic shock, pneumonia, skin and soft tissue infection (Ryu et al., 2014; Malik et al., 2018). Chitosan nanoparticles were found to exhibit strong antibacterial activity compared to those achieved simultaneously with the solution alone. Chitosan, a versatile hydrophilic polysaccharide derived from chitin, has a broad antimicrobial spectrum to which gram-negative, gram-positive bacteria and fungi are highly susceptible (Jana, Jana, 2020). Moreover, the antimicrobial activity of designed nanoparticles was significantly higher for S. aureus than P. aeruginosa. Previous studies also revealed remarkable susceptibility of rhubarb extracts to gram positive bacteria (S. aureus) than gram negative bacteria (such as E. coli and P. aeruginosa) (Sayyahi et al., 2020; Tegos et al., 2002). Recently, bactericidal effect of four herbal extracts (Rhubarb, Scutellaria Root, Phellodendron Bark, and Coptidis Rhizome) was evaluated on different bacterial strains and it was observed that the effect of herbs on Gram-positive bacteria is better compared to Gram-negative bacteria (Wu et al., 2021). The cell walls of gram-positive bacteria are more sensitive to many antimicrobial compounds than gram negative bacteria where inherent tolerance can be the contributing factor (Koohsari et al., 2015). Chitosan incorporation as a nano-polymer significantly enhances antibacterial activity of rhubarb. Nanoparticles are able to deliver their content directly into the cell cytoplasm owing to their small size by penetration into the plasma membrane, having a special advantage in terms of intracellular transport and hence contributes to antibacterial effectiveness.

Significant wound healing was observed with gel formulation in rats when compared with standard and control. Gel formulation healed wounds within 6 days, whereas with solution and standard wound contraction was found to be 91.7% and 79%, respectively after 8^th day of treatment. However, 100% wound contraction with standard and solution was observed after the 10^th day.

Our findings lead to a new formulation that can treat wound effectively. The synergistic herbal formulation can also be used in the topical application for treating a group of medical condition consisting of wounds, cutaneous abscesses, pimples, skin infections, burns and skin pigmentation. The formulation opens new avenues for wound healing that will offer better patient care and more efficient development. In fact, the formulation developed using herbal combination is new in the prior art that could shed light on novel options in the limited world literatures.

CONCLUSION

Novel herbal compositions containing rhubarb in chitosan nanoparticles loaded in a gel formulation for enhanced wound healing and other medical conditions was developed successfully. Nanoparticles were successfully optimized by DESIGN EXPERT. The results of a box-behnken design revealed that the polymer, cross-linker and the drug concentration significantly affected the dependent variables i.e. particle size, zeta potential, drug entrapment and % drug release. Hydrogel based formulation was developed using combination of polyvinylpyrrolidone and carboxymethylcellulose. Antimicrobial and wound healing activity of gel formulation performed on rats exhibited a significant zone of inhibition and wound contraction when compared with control and marketed formulations.

ACKNOWLEDGEMENT

The authors extend their appreciation to Taif University, Saudi Arabia, for supporting this work through project number: TU-DSPP-2024-131.

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Edited by

• Associated Editor:
  Skylar Carlson.

Publication Dates

• Publication in this collection
  20 Jan 2025
• Date of issue
  2025

History

• Received
  25 Aug 2023
• Accepted
  15 Mar 2024