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carboxymethyl tamarind seed polysaccharide loaded with moxifloxacin beads for effective wound heal
International Journal of Biological Macromolecules 140 (2019) 1106–1115
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Spongy wound dressing of pectin/carboxymethyl tamarind seed polysaccharide loaded with moxifloxacin beads for effective wound heal Ashlesha P. Pandit a,⁎, Kanchan R. Koyate a, Ashwini S. Kedar a, Vaishali M. Mute b a b
Department of Pharmaceutics, JSPM Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India Department of Pharmacology, JSPM Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 25 April 2019 Received in revised form 22 August 2019 Accepted 23 August 2019 Available online 27 August 2019
a b s t r a c t An attempt was made to formulate moxifloxacin loaded alginate beads incorporated into spongy wound dressing to heal chronic wounds as well as to reduce frequency of painful dressing change. Moxifloxacin loaded beads (sodium alginate:pectin, 1:1) were prepared by ionic gelation method, with entrapment efficiency 94.52%, crushing strength 25.30 N and drug release 90.52%. Beads were further incorporated into wound dressing, made of pectin and carboxymethyl tamarind seed polysaccharide (CMTSP). Spongy wound dressing was obtained by freeze drying technology, which showed good folding endurance, high wound fluid absorption and good crushing strength. Drug release was found to be 85.09%. Dressing made of CMTSP:pectin (1.5:2) showed good water vapour transmission and antibacterial activity. Porous nature of dressing absorbed exudates of wound. Excision wound model in rats revealed wound healing within 17 days: groups I (control), II (moxifloxacin beads loaded wound dressing), III (moxifloxacin beads), IV (pectin film) and V (sodium alginate film) showed 65.28, 99.09, 86.90, 66.84 and 64.30% wound closure, respectively. To conclude, moxifloxacin beads loaded spongy wound dressing has good healing and wound closing potential compared to pectin film and moxifloxacin beads. Thus, the formulation is novel for biomedical application which reduced the frequency of painful dressing change. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Wound healing is a major health care challenge. With an unparalleled increase in incidence of chronic wounds at accidents, traumatic wounds and complicated post-surgical scenario, it is highly desirable to design a high performance antibacterial wound-care system [1]. A wound can be defined as an injury or disruption to the skin and can extend to subcutaneous tissue, muscles, tendons, nerves, vessels, and even to the bone [2]. Based on the nature and repair process wounds can be classified as chronic and acute wound. However, chronic wounds are more prone to contamination and usually involve significant tissue loss that can affect vital structures such as bones, joints, and nerves [3]. To overcome this problem, wound dressings are used to protect the wound from external environment and contamination. Topical bioactive agents like solutions, creams, and ointments are also used to cure wound, but are less effective due to rapid fluid absorption, thus become mobile losing rheological characteristics [4]. Therefore, solid wound dressings are preferred in the case of exudate wounds, due to better exudate management and prolonged residence at the wound site than other formulations. Currently, advanced dressings of bioactive agents ⁎ Corresponding author at: JSPM Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India. E-mail address: [email protected] (A.P. Pandit).
https://doi.org/10.1016/j.ijbiomac.2019.08.202 0141-8130/© 2019 Elsevier B.V. All rights reserved.
are used to get faster wound healing [5]. However, porous wound dressings are treatment of choice as these dressings absorb wound fluid exudates and also allow water vapour transmission [6]. Moreover, wound dressing efficiency is enhanced by loading beads into it. These beads are loaded with antibacterial drug to sustain release of drug and are prepared by ionic gelation method. Antibiotics are of choice to treat wound infection [7]. However, one of them is moxifloxacin, for prevention and elimination of microorganisms at wound sites. Moxifloxacin (MX) is a fourth-generation fluoroquinolone antibiotic, with similar in vitro activity as that of ciprofloxacin and ofloxacin against Gram-negative bacteria, but enhanced activity against Gram-positive bacteria [8]. Moxifloxacin is beneficial for topical treatment of infected wound as it accelerates wound repair process [9]. Pectin is a natural prophylactic substance used for wound healing which acts against poisoning with toxic cations. Pectin has styptic and curing effects which are well documented in healing ointments [10]. However, due to its poor intrinsic mechanical properties, it is used typically in conjunction with other polymers. Extensive literature revealed that researchers developed a ternary nano dressing consisted of titanium dioxide nano particle loaded chitosan-pectin [11]. Beneficial effects of beads in wound care has been studied thoroughly as follows: alginate beads containing the polar lipid monoolein to manage wet wounds by providing improved uptake of excess exudate while releasing adenosine locally for promotion of healing [12]; calcium-alginate beads to immobilize tetracycline
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and improve an antibacterial biomaterial for open wound [13]; new composites based on Ca-alginate hydrogels released activated charcoal (AC) particles with adsorbed povidone iodine (PVP-I) as a model antimicrobial substance in a physiological-like environment [14]. Recently, newer approach is the use of antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility of wound dressing for joints skin wound healing [15] and novel antibacterial anti-oxidant electro-active injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing [16]. Currently, wound dressings are of choice to heal wound. However, changing of dressing is uncomfortable to the patients. Time required to heal wound reduces their quality of life. Dressing changes also represent a major financial and logistical burden to patients. Keeping in view the above problems, dressing change frequency was planned to reduce by using biomaterials such as pectin, carboxymethyl cellulose and alginate which themselves have wound healing potential. Thus, the aim was to formulate porous wound dressing embedded with moxifloxacin-alginate beads. Dressing was made of combination of pectin and carboxymethyl tamarind seed polysaccharide for better wound care. Application of this novel dressing was planned to reduce the frequency as well as pain which occurred during dressing change, each time. Beads loaded dressing allowed slow release of MX at the wound area. Advanced treatment modality of freeze dried technology thought to make the dressing spongy which helped to absorb exudates of wound and permeated water vapour transmission, required for fast wound heal. 2. Materials and methods 2.1. Materials MX was received as gift sample from Macleods Pharmaceuticals, India. Pectin, sodium alginate and nutrient agar were procured from Hi-media, Mumbai, India. Carboxymethyl tamarind seed polysaccharide (CMTSP) was kindly gifted by Alfa Exim, India. 2.2. Formulation of MX beads Beads were prepared by ionic gelation method using sodium alginate and pectin [17]. Sodium alginate: pectin ratios 1:1 (B1), 1:1.5 (B2), and 1:2 (B3) were added in distilled water (20 ml) and stirred for 15 min. MX (500 mg) was added to the above solution and again stirred. Polymer solution thus obtained was added drop by drop using syringe to the calcium chloride (20%) solution to get spherical beads. Beads were filtered and washed three times with distilled water [18]. 2.3. Entrapment efficiency of beads Entrapment efficiency of MX-loaded beads (MX-B) is the total amount of drug present in the product. Beads were washed with distilled water and were analyzed to calculate the amount of free drug. Entrapment efficiency was calculated using Eq. (1) [19]: Entrapment Efficiency ð%Þ ¼ Total drug added−Free drug=Total drug added 100
ð1Þ
2.4. Crushing strength of beads Crushing strength is the maximal force corresponding to the peak of the force-time plot. Crushing strength of the beads was determined using a CT3 Texture Analyzer (Brookfield Engineering Laboratories, Inc., USA), operating at 0.05 N load cell. Thereafter, force (N) vs time (s) plot was recorded using texture analyzer software. In short, a spherical bead was placed on lower flat plate. The upper punch of 25.4 mm
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diameter and 35 mm length, made of clear acrylic material, was moved down, slowly at a constant rate of 0.3 mm/s [20] (Texture Pro CT). 2.5. Antibacterial activity Antibacterial activity of MX (MX 500 μg dissolved in 0.5 ml DMSO) as well as MX-loaded beads (equivalent to 500 μg MX) against causative microorganisms of wound infection S. aureus [21] and P. aeruginosa was evaluated using agar diffusion method [8]. Herein, sterile nutrient agar media was poured into sterilized plate under a laminar airflow unit. Thereafter, diluted suspension of microbe (standardized concentration of 108 cells/ml) was spread onto filled agar plate and was kept for solidification. Next, cups were punched on agar plate and filled with solution or beads, separately. Petri plates were then incubated at 37 °C for 24 h (n = 3). Lastly, the zone of inhibition was measured and reported as mean ± S.D. [22]. 2.6. In-vitro drug release of beads In vitro release of MX-B was performed using modified USP dissolution test apparatus (Basket type, TDT 08, Electro lab, India). Initially, beakers (250 ml) were placed in dissolution flasks of the dissolution tester and were filled with phosphate buffer solution pH 7.4 (100 ml). An accurately measured amount of beads, equivalent to 500 μg MX, were filled in baskets and suspended in beakers containing dissolution media. Thereafter, the apparatus was run at 150 rpm at 37 ± 0.5 °C. Further, aliquots of samples (5 ml) were collected till 8 h and analyzed by UV spectrophotometer at λmax 292 nm. Sink condition was maintained in dissolution medium by replacing aliquots each time with same volume at 37 ± 0.5 °C [6,23]. 2.7. Scanning electron microscopy Morphology and surface topography of bead was studied using SEM. Prepared beads were coated with gold–palladium under an argon atmosphere at room temperature and then the surface morphology of the beads was studied by scanning electron microscopy at 50× and 500× magnification (JEOL Oxford). 2.8. Formulation of beads loaded wound dressing Porous wound dressing of MX-loaded beads (MX-BWD) was prepared by freeze drying method [24]. Briefly, aqueous polymer blends of CMTSP (1.0 and 1.5 g): pectin (1.0 and 2.0 g) and glycerin (1 ml) were homogenized for 15 min (Table 1). Polymeric solution (2 ml), thus formed, was poured into glass mold (dimensions: 3 × 3 cm) and weighed amount of beads (equivalent to single dose of 500 μg) were uniformly spread into the mold. Further, beads were covered by adding remaining 2 ml of polymer solution. The resulting solution was then subjected to freeze drying technology (Martin Christ, Germany, Alpha 1–2 LD Plus) at −42.5 °C for 48 h to get porous mass [25]. The obtained spongy wound dressing (F1 to F4) was stored until further use. 2.9. Characterization of beads loaded wound dressing Wound dressing was characterized for thickness using micrometer screw gauge. The pH of dressing was determined by immersing MXBWD in normal saline solution till it reached equilibrium. Dressings were then removed from the solution and pH of the solution was determined using digital pH meter. All tests were performed in triplicate and average values were recorded [26]. Next, the folding endurance of dressing was measured manually. It is the number of times the patch is folded at the same place to break the patch. It provides an indication of brittleness [27]. Tensile strength was studied using texture analyzer (CT3 Texture Analyzer, Brookfield Engineering Labs. Inc., USA) with
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Table 1 Formulation of beads loaded spongy film dressing. Batch
MX (μg)
CMTSP (g)
Pectin (g)
Thickness (mm)
pH
Folding endurance
Tensile strength (N)
Wound exudate absorptive capacity (%)
Drug release (%)
F1 F2 F3 F4
500 500 500 500
1.0 1.0 1.5 1.5
1. 0 2.0 1.0 2.0
0.41 ± 0.012 0.47 ± 0.01 0.44 ± 0.012 0.5 ± 0.015
5.87 5.42 6.16 5.26
148 ± 4 164 ± 12 143 ± 7 161 ± 4
8.87 ± 1.1 11.17 ± 1.2 9.16 ± 0.77 15.88 ± 0.65
683.99 ± 3.28 606.89 ± 4.37 578.63 ± 4.72 552.07 ± 2.89
85.21 ± 2.11 80.83 ± 0.89 85.62 ± 1.09 85.09 ± 2.03
TexturePro CT software. Dressing (2 × 5 cm) was cut and fixed between two clamps of texture analyzer with a load of 50 N. The attached strip was then pulled at the rate of 10 mm/min. Tensile strength at break of dressing was calculated by following Eq. (2): Tensile strength ¼ ðbreaking forceÞ= cross−sectional area N=m2
ð2Þ
2.10. In-vitro drug release of MX-BWD Wound dressings were immersed in beakers (capacity 500 ml) containing 100 ml phosphate buffer solution (pH 7.4) at USP dissolution test apparatus Type II (paddle type) [18], at 37 ± 0.5 °C at 50 rpm. At predetermined time, aliquot of 5 ml was withdrawn at an interval of 1 h till 10 h and replaced with same volume of fresh phosphate buffer solution. Then, the volume of samples were diluted up to 10 ml and analyzed by UV spectroscopy at 292 nm [8]. 2.11. Wound exudates absorptive capacity Fluid absorption capacity of wound dressings was measured by using simulated wound fluid (SWF). SWF was prepared by dissolving 0.02 M calcium chloride, 0.4 M sodium chloride, 0.08 M methylamine, and 2% w/v of bovine serum albumin in distilled water (100 ml) at pH 7.4. The previously weighed dressing (3× 3 cm) was dipped in SWF. After, fixed time interval, swollen dressing was wiped with cotton to remove excess SWF on the surface, and re-weighed. This was repeated until a constant weight was obtained. Thereafter, change in the weight of film was noted [28,29]. Wound exudates absorptive capacity (%) was calculated using Eq. (3): Wound exudates absorptive capacity ð%Þ ¼ ðWs−WdÞ=Wd 100 ð3Þ where Ws and Wd are weight of the swollen and dry film, respectively. 2.12. Water vapour transmission Water vapour transmission rate was determined by fixing MX-BWD (20 mm diameter) to Franz diffusion cell containing distilled water (20 ml). An open cell was used as a control. A desiccator, was kept in a hot air oven at 35 °C. Diffusion cell was placed in desiccator, which was further re-weighed at periodic intervals (1, 3, 5, 7, 9, 12, 24 h). Here, weight loss corresponds to evaporated water content. Thereafter, water vapour transmission rate was calculated using following Eq. (4) [26,30]: Water vapour transmission ¼ ml 24=tS
ð4Þ
where ml is mass loss during the time t (measured in h) and S is the area of cell mouth.
2.14. Degradability study of dressing Wound dressing was subjected to degradation study based on the earlier reported method [21]. Briefly, dressings were weighed (900 mg, 2 × 2 cm) and immersed in 20 mL SWF medium (pH 7.4) with continuous shaking at 100 rpm at 37 °C. Initial weight of the dressing was noted as Wi. Each set of dressings were removed at time interval of 7, 14, 21 days from the medium, washed with deionized water to clean ions adsorbed on surface and dried in an oven at 50 °C for 48 h and then reweighed (Wt). The degradation of dressing was calculated as follows: Degradation ð%Þ ¼ ½ðWi−WtÞ=Wi 100
ð5Þ
2.15. In-vivo wound healing study Healthy male Wistar albino rats (60 days old), weighing between 150 and 200 g were acclimatized to the laboratory environment for a period of 3 days. Animals were housed at temperature 22 ± 1 °C and humidity 35 ± 5% to 65 ± 5% RH in clean sterile polycarbonate cages containing autoclaved paddy husk, with free access to water and food, and kept under 12/12 h light/dark cycles. All the experimental procedures and protocol were approved by the Institutional Animal Ethics Committee, India, constituted under Committee for Purpose of Control and Supervision of Experiments on Animals (IAEC-16-011). Animals were divided into five groups containing 3 animals in each group. Group I: control (CG); Group II: moxifloxacin beads loaded in spongy wound dressing (MX-BWD); Group III: moxifloxacin beads (MX-B); Group IV: pectin film (PF); and Group V: sodium alginate film (SAF). 2.16. Excision wound model Animals were anaesthetized with anaesthetic ether by open mask method and shaved on both sides of back with electric clipper. Thereafter, excision wound was inflicted on the dorsal thoracic region 1–1.5 cm away from the vertebral column and 5 cm away from ear of each animal. A full thickness excision wound of circular area 100 mm2 and 2 mm in depth was created along the markings. The entire wound was left open. Animals were closely observed for any infection and those which showed signs of infection were separated and excluded from the study. Spongy dressing was applied on the wound once, on day 0 to cover the wound [31,32]. Wound areas were measured planimetrically on days 1, 7, 14, and 17 and percent wound closure was calculated using Eq. (6): Wound closure ð%Þ ¼ Wound area on 0 day−Wound area on nth day Wound area on 0 day 100
ð6Þ
where, n is the number of days (1, 7, 14 and 17). 2.17. Histological study
2.13. Antibacterial activity Antibacterial activities of (a) pectin and CMTSP against P. aeruginosa, (b) MX-BWD against P. aeruginosa, (c) pectin and CMTSP against S. aureus, (d) MX- BWD against S. aureus were performed as discussed previously, in Section 2.5. The zone of inhibition was measured in mm [13].
Rats were sacrificed on day 17 after complete wound heal. Next, wounded area of skin, containing dermis and hypodermis, was isolated and carefully trimmed with cutter. It was then fixed in 10% neutral formalin solution. After paraffin embedding, 3 to 4 μm sections were stained with hematoxylin and eosin (H and E) for the study of tissue appearance. All the slides were observed under light microscopy [11].
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2.18. Statistical analysis
3.1. Entrapment efficiency
Experimental results were expressed as the mean standard error using software GraphPad Prism (Version 5, GraphPad Software Inc., La Jolla, CA). The results were analyzed statistically using two-way analysis of variance (ANOVA) followed by Bonferroni post-tests, where P b 0.001 was considered as statistically significant [32].
The amount of increased pectin concentration (1.0 to 2.0 g) resulted in decreased entrapment efficiency of drug (94.52 ± 1.5, 93.56 ± 1.3 and 89.83 ± 2.1%). Calcium ions induce chain–chain associations that formed junction zones (called egg-box-junctions) which was responsible for gel formation by both sodium alginate and pectin. The chemical interactions occur between calcium ions and guluronate and galacturonate blocks present in sodium alginate and pectin, respectively. Thus, increase in pectin concentration, decreased an entrapment efficiency. The obtained result is in agreement with previous results [19].
2.19. Cell compatibility of wound dressing Cell compatibility of wound dressing was studied based on the mechanism of angiogenesis. Angiogenesis is a complex physiological process required for healing the wounds, which involves restoring blood flow to tissues after injury, has become a major focus of study for wound biologists. Angiogenic activity of wound dressing was studied using an in vitro chick chorioallantoic membrane (CAM) model [33]. The CAM is a vascular extra-embryonic membrane found in eggs of some amniotes, such as chick. It is formed on day 4 of incubation. Briefly, nine days old fertilized chick eggs were selected. A small window in the egg shells was opened, carefully. Then, a sterile wound dressing (2 × 2 cm) was positioned at the joint of two large blood vessels. The window was resealed with adhesive tape, cautiously and the eggs were incubated further at 37 ± 1 °C in a well-humidified chamber. Next, the tape was opened after 72 h to observe new blood vessel formation. The results were compared with the control eggs without the dressing.
2.20. Stability study Stability study of wound dressing was studied to obtain a stable product which assures safety and efficacy, till shelf life, at defined storage and package conditions. Accelerated stability study was performed according to ICH guidelines to assess the combined effect of excipients of beads and dressing on stability of the dressing [23]. Dressing was packed in amber color bottle, sealed, and kept at stability chamber (Thermolab, India) at 40 ± 2 °C/75 ± 5% RH. The samples were evaluated for pH, folding endurance, microbial growth, strength, entrapment efficiency and drug release after 7 and 15 days and 1, 3, and 6 months.
3. Results and discussion MX-B were formulated by using sodium alginate and pectin by ionic gelation method. Here, sodium alginate forms a viscous solution with water, while in contact with calcium chloride solution forms spherical beads.
3.2. Crushing strength Beads exhibited the maximal crushing strength at the highest amount of pectin (25.30, 25.52 and 26.36 N) (Fig. 1a). Crushing strength increased with increased amount of pectin. Pectin provides good strength to beads. Pectin, also known as pectic polysaccharides, is rich in galacturonic acid. It contains several distinct polysaccharides such as homogalacturonans which is linear chains of α-(1-4)-linked Dgalacturonic acid. Therefore, high amount of pectin provides good strength and rigidity to the beads [34]. 3.3. Antibacterial study Antimicrobial activity of MX and MX-B against S. aureus was 57.04 ± 0.65 mm and 56.02 ± 0.5 mm, respectively, and 59.4 ± 0.55 mm and 58.6 ± 0.43 mm, respectively against P. aeruginosa (Fig. 2a–d). The release of MX through beads in the media was delayed due to presence of pectin. Therefore, beads showed, comparatively, less zone of inhibition than MX. Thus, MX-B were potential candidate for effective wound dressing. 3.4. In vitro drug release study of MX-B Increase in concentration of pectin decreased the drug release from the beads MX-B. Herein, formulation B1 (1:1), B2 (1:1.5) and B3 (1:2) with increased concentration of pectin showed 23.40 ± 1.3, 22.41 ± 2.1 and 20.81 ± 1.8%, respectively, drug release in first 2 h; 47.67 ± 1.5, 42.48 ± 2.4 and 41.18 ± 2.2% release within 4 h; 69.39 ± 1.7, 68.10 ± 2.4 and 65.39 ± 1.1% release within 6 h; followed by 90.54 ± 1.6, 88.24 ± 1.5 and 87.68 ± 2.7% release, respectively in further 8 h (Fig. 3). Beads B1 and B2 at lower concentration of pectin released the drug rapidly than B3. However, presence of more amount of pectin at B3 forms thick layer on the surface of beads. Pectin gets swell in contact with dissolution media, thus delayed the release of MX than B1 and B2 due to slow dissolution of drug [7,35]. Here, all batches exhibited
Fig. 1. (a) Crushing strength of beads; tensile strength of (b) F4.
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Fig. 2. Antibacterial activity of (a) moxifloxacin against P. aeruginosa, (b) moxifloxacin beads against P. aeruginosa, (c) moxifloxacin against S. aureus, (d) moxifloxacin beads S. aureus.
linear release. However, at high amount of pectin, the behavior of release of MX though beads could be non-linear.
helped to enhance the wound exudates absorptive capacity [24]. Also, the wound exudate enters in spongy dressing and provides medium to release drug from beads.
3.5. Selection of batch of beads 3.8. Characterization of MX-BWD Beads B1 (1:1), B2 (1:1.5) and B3 (1:2) showed entrapment efficiency of 94.52 ± 1.5, 93.56 ± 1.3 and 89.83 ± 2.1%, respectively, within 8 h. Increase in pectin concentration decreased the entrapment efficiency. The crushing strength of beads was not much affected for B1 (25.30 N) and B2 (25.52 N), although, found to be increased in B3 (26.13 N). The release of MX through B1, B2 and B3 was 90.52, 88.47 and 88.23%, respectively, within 8 h. The effect of pectin in sodium alginate: pectin beads exhibited results as follows: highest amount of drug was entrapped in beads B1 with drug release within 8 h with good crushing strength, while fewer amount of drug was entrapped in B2 and B3. Therefore, formulation B1 was selected for further studies. 3.6. Scanning electron microscopy (SEM) SEM of the beads revealed spherical structure (Fig. 4a) with rough surface. Wherein, spherical nature of beads was required to load the beads in the dressing. Similar result was obtained in earlier study for sodium alginate beads which exhibited rugged surface [13]. 3.7. Formulation of MX-BWD Wound dressing is an advanced technology, used for treatment of acute as well as chronic wounds. Herein, MX-B were prepared by ionic gelation method using sodium alginate and pectin. Beads were loaded into dressing base consisted of pectin and CMTSP (Fig. 4b). Next, dressing was made spongy by freeze drying method (Fig. 4c). Spongy nature of dressing imparted flexibility in movements to the patient as well as
Dressing thickness measurement is often vitally important for absorbing the exudate of wound in the spongy mass. Thickness of MXBWD varied in the range of 0.41 ± 0.012 to 0.5 ± 0.015 mm. F1 dressing was less thick, while F4 showed highest thickness amongst all the formulations. Thick wound dressing is important parameter to provide sufficient strength as well as impart spongy nature [26]. High folding endurance of dressing is the indication of flexibility and elasticity that allows the free movements of body parts after application [26]. F2 exhibited highest folding endurance at 163 ± 12, while F3 showed lowest at 143 ± 7. High concentration of pectin showed more flexibility and therefore, depicted the highest folding endurance [28]. Tensile strength testing provides an indication of the strength and elasticity of the dressing. It is a versatile tool for the mechanical characterization of pharmaceutical film dressings. F4 dressing exhibited the highest tensile strength at 15.88 ± 0.65 N (Fig. 1b). The addition of higher amount of pectin increased the tensile strength of the dressing. F1 shows the tensile strength of 8.87 ± 1.1 N. High tensile strength is always favored that prevents the breaking of dressing during application, storage and transportation [35]. Wound dressing shows the pH in the range between 5.26 and 6.16. These values are in close agreement with the dressing pH values (5.25–7.90) reported earlier [26]. Dressing pH is a vital parameter for wound dressing, which favors to regulate infection at wound surface as well as accelerates the formation of fibroblast proliferation. The pH of normal human skin ranges between 4.0 and 6.8, therefore, pH of wound dressing should be slightly acidic at wound surface, which thus helps to accelerate the wound heal. An alkaline wound
Fig. 3. Drug release through beads.
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Fig. 4. Scanning electron microscopy of moxifloxacin beads (a) 50× magnification, (b) distribution of beads in wound dressing (c) porous wound dressing.
pH is associated with reduced wound healing process. Open wounds characteristically have a neutral to alkaline pH that ranges 6.5 to 8.5, while chronic wounds 7.2 to 8.9. An alkaline wound environment impairs the healing and immunological response by promoting bacterial growth increasing proteolytic activity, inhibiting fibroblasts and decreasing oxygen supply. 3.9. In-vitro drug release Wound dressing F2 and F4 with high amount of pectin resulted in 37.27 and 41.45% drug release, respectively, within first 5 h, followed by 80.83 and 85.09% drug, respectively within 10 h. Formulation F1 and F3 showed 42.37 and 39.16% within first 5 h, followed by 85.21 and 85.62% drug release, respectively within 10 h as shown in Fig. 5. Thus, it was observed that increased concentration level of CMTSP delayed the drug release from the wound dressing and resulted in slow release of drug from the wound dressing compared to other formulations [24]. However, drug release behaves differently at high levels of CMTSP. The release of MX through dressing crosses the swollen dressing due to CMTSP as well as embedded beads in it. This resulted in comparatively delayed MX release through dressing than beads. 3.10. Wound exudates absorptive capacity Wound exudates absorption capacity of dressing was due to two processes: water absorbed by materials and water being retained in
the porous structure. Dressings absorbed 483.99 ± 0.28 to 606.89 ± 0.37% of SWF within 60 min. Fast absorption of exudates into dressing is vital for fast wound healing, because presence of high amount of exudate slows down cell proliferation. Moreover, wound exudate retained at porous wound dressing provides media to release drug from beads. Wound exudates absorption capacity of dressing was found to be directly proportional amount of pectin due to its water retention capacity. High wound exudates absorptive capacity indicated structural integrity of the dressing [35,36].
3.11. Water vapour transmission Moisture regulation of wound is an important aspect and results in getting wound conditions more amenable to healing. The dried wounds can delay the healing process, while accumulation of exudates influences the occurrence of the infection. Therefore, capacity to exchange the moisture through the dressing at optimum level is required to fast heal the wound. Evaporative water loss for normal skin is 204 ± 12 g/m2/day; while for injured skin range from 279 ± 26 g/m2/day to 5138 ± 202 g/m2/day for a granulating wound. Herein, an adequate level of moisture, is required to avoid risk of wound dehydration as well as buildup of exudates on the wound, water vapour transmission rate from injured wound should be in the range 2000–2500 g/m2/day [30]. The prepared MX-BWD (F4) showed weight loss and water vapour transmission rate of 4.15 and 31.71 ± 18 g/m2/day, respectively after 1 h; 7.82 g and 59.77 ± 19 g/m2/day, respectively after 3 h; 12.14 g
Fig. 5. Drug release through wound dressing.
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Fig. 6. Antibacterial activity of (a) pectin and CMTSP against P. aeruginosa, (b) wound dressing against P. aeruginosa, (c) pectin and CMTSP against S. aureus, (d) wound dressing against S. aureus.
and 92.78 ± 27 g/m2/day, respectively after 5 h; 15.73 and 120.22 ± 25 g/m2/day, respectively after 7 h; 17.61 g and 134.59 ± 46 g/m2/day, respectively after 9 h; 26.98 g and 201.63 ± 49 g/m2/day, respectively after 12 h; 42.24 g and 322.85 ± 51 g/m2/day after 24 h, thus, close to the adequate range of the ideal wound dressing. Such water vapour transmission rate can strengthen the cellular re-epithelialization. Dressing F4 showed good water vapour transmission rate because of the highest concentration of both polymers [27]. Porous structure of wound dressing allowed water vapour transmission to a great extent which favors fast wound heal [7]. Decreased oxygen tension lowers the pH which affects angiogenesis, immunological activity and collagen formation. 3.12. Antibacterial activity CMTSP did not show zone of inhibition against both P. aeruginosa and S. aureus, while wound dressing exhibited nearly same zone as that of beads. Zone of inhibition of pectin and wound dressing against P. aeruginosa was 13.2 ± 0.3 and 57.8 ± 0.32, respectively (Fig. 6a and b). While, zone of inhibition of pectin, CMTSP and wound dressing against S. aureus was 11.8 ± 0.2, 12.2 ± 0.4 and 55.9 ± 0.45 mm, respectively (Fig. 6c and d). MX-BWD was effective against S. aureus and P. aeruginosa (zone of inhibition 55.9 ± 0.45 mm and 57.8 ± 0.32 mm, respectively). Wound dressing showed same zone of inhibition against both the Gram positive and Gram negative strains of bacteria as that of MX-B. Therefore, wound dressing was considered to have good antibacterial activity. 3.13. Degradability study of dressing Wound dressings degraded 8–11% within 7 days, 18–23% within 14 days and 28–38% within 21 days. Pectin has potential to bind to CMTSP and protect the dressing from degradation. Also, higher swelling and porosity account for increased biodegradation. MX was released within 10 h while porous dressing remained at the wound area till degradation. Thus, the above results accurately forecasted degradation profile of the dressing. The degradable characteristic would contribute to the reduced frequency of wound dressing change.
3.14. In-vivo study (excision wound model) Wound healing activity of the wound dressing was studied in the rat using excision wound model. Wound closure (%) of Group I: control (CG); Group II: F4 (MX-BWD); Group III: B1 (MX-B); Group IV: (PF); Group V: (SAF) on days 1, 7, 14 and 17 are as shown in Table 2 and Fig. 7. Comparison between Group I (CG) vs Group II (MX-BWD) of excision wound model using Two-way ANOVA is as follows: On day 1, MX-BWD showed 11.81% and CG showed 6.61% wound closure. It was found statistically significant with P˂0.001. On day 7, MX-BWD showed 42.20% and CG showed 16.44% wound closure. CG and MXBWD showed 25.76% difference in wound closure. It was found statistically significant (P ˂ 0.001). On day 14, MX-BWD showed 90.90% and CG showed 46.28% wound closure. It was found statistically significant (P ˂ 0.001). On day 17, MX-BWD and CG showed 99.09% and 65.28% wound closure, respectively. It was found statistically significant (P ˂ 0.001). PF showed 7.07% on day 1, 20.20% on day 7, 54.65% on day 14 and 66.84% on day 17 wound closure. SAF showed 6.59% on day 1, 27.17% on day 7, 47.82% on day 14 and 64.30% on day 17 wound closure. Group III (MX-B) showed 7.21% on day 1, 24.74% on day 7, 67.01% on day 14, and 86.59% on day 17 wound closure. MX-BWD treated rats, thus, showed faster healing within 17 days than MX-B loaded dressing, while PF and SAF showed very less healing of wound [1,31,32].
3.15. Histopathological study At the end of experiment, on day 17, rats of Group I (control) showed formation of scar (represented by arrow in Fig. 8a), severe inflammatory cell infiltration and fibroblast proliferation (represented by circle in Fig. 8a). Moreover, rats of Group II (MX-BWD) showed re-epithelialization (represented by arrow in Fig. 8b), and neovascularization (represented by small arrow in Fig. 8b) and regeneration of hair follicles (represented by circle in Fig. 8b). However, rats of Group III (MX-B) revealed formation of scar (represented by arrow in Fig. 8c), minimal inflammatory cell infiltration (represented by circle in Fig. 8c) and neovascularization (represented by small arrow in Fig. 8c) and healing under scar. Thereafter, rats of Group IV (PF) showed minimal formation of scar
Table 2 Rate of wound closure within seventeen days. Groups
Wound closure (%) on day 1 (Mean ± SD)
Wound closure (%) on day 7 (Mean ± SD)
Wound closure (%) on day 14 (Mean ± SD)
Wound closure (%) on day 17 (Mean ± SD)
Group I Group II Group III Group IV Group V
6.61 ± 0.72 11.81 ± 0.74 7.21 ± 0.58 7.07 ± 1.1 6.59 ± 0.79
16.44 ± 0.67 42.2 ± 1.1 24.74 ± 0.32 20.20 ± 0.75 27.17 ± 1.34
46.28 ± 0.55 90.90 ± 0.63 67.01 ± 0.175 54.65 ± 0.83 47.82 ± 0.72
65.28 ± 0.43 99.09 ± 0.76 86.59 ± 0.21 66.84 ± 0.64 64.30 ± 1.15
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Fig. 7. Comparative study of wound healing (excision wound model) in rats consist of five groups over a period of 17 days treatment. Values are ±SD of n = 5. Two-way ANOVA was used for statistical analysis. #P N 0.05. Group I: control; Group II: moxifloxacin beads loaded in wound dressing; Group III: moxifloxacin beads; Group IV: pectin film; Group V: sodium alginate film. Scale bar 5 mm.
tissue (represented by arrow in Fig. 8d). Rats of Group V (SAF) showed normal histological structure of wound (represented by Fig. 8e) [32]. Thus, the dressing MX-BWD exhibited rapid wound healing with closing of wound.
3.16. Cell compatibility of wound dressing A complex process of wound closing involves angiogenesis required for wound healing where its induction is beneficial in many clinical
Fig. 8. Histopathological study of wound tissue after 17 days, where (a) Group I (control), (b) Group II (moxifloxacin beads loaded wound dressing), (c) Group III (moxifloxacin beads), (d) Group IV (pectin film), (e) Group V (sodium alginate film). Scale bar 10 μm.
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References
Fig. 9. Cell compatibility of wound dressing using CAM model (a) control (b) dressing.
situations for achieving wound closure. Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels, formed in the earlier stage of vasculogenesis. During this process, proliferating capillaries bring oxygen and micronutrients to growing tissues and remove catabolic waste products. This leads to angiogenesis in wounds. In the chick CAM model, dressing showed an increase in density of blood capillaries on the treated membrane surface (Fig. 9a) compared to control group (Fig. 9b) of eggs. Thus, results indicated good angiogenic activity of dressing. Again, angiogenesis during wound repair serves the dual function of providing the nutrients demanded by the healing tissues and contributing structural repair through the formation of granulation tissue. 3.17. Stability study No significant change was observed at dressing F4 for pH (5.28), tensile strength (15.61 ± 0.43), folding endurance (160 ± 9), and drug release (84.98 ± 2.14) at 40 ± 0.5 °C/75 ± 5% RH till 6 months. At visual examination, the microbial growth was not observed on the surface of dressing. 4. Conclusion Wound healing is a major health care challenge. Dressing changes are uncomfortable for patients, which also causes a major financial and logistical burden. Therefore, an attempt was made to formulate moxifloxacin-loaded beads incorporated into spongy wound dressing with an objective to heal chronic wounds to reduce dressing change frequency. Dressing allowed slow release of drug through beads at wound area. Spongy dressing absorbed wound exudates and permeated water vapour transmission, required for fast heal. Thus, porous dressing delivered moxifloxacin on surface of wound with slow release, while preventing its penetration inside. Novel wound dressing achieved more contact time with wound area, easy applicability, less pain to the patient due to reduce frequency of dressing change. Ease of applicability at the wound area without any pain; reduced frequency of changing wound dressing with less pain to patients, ultimately, avoiding the chances of amputation, is major breakthrough at wound care system. Declaration of Competing Interest Authors declare that there are no conflicts of interest. Acknowledgement Authors are grateful to Macleods Pharmaceuticals, India for gifting moxifloxacin and Alfa Exim, India providing gift sample of CMTSP.
[1] J. Boateng, O. Catanzano, Advanced therapeutic dressings for effective wound healing—a review, J. Pharm. Sci. 104 (2015) 3653–3680, https://doi.org/10.1002/ jps.24610. [2] P.G. Bowler, B.I. Duerden, D.G. Armstrong, Wound microbiology and associated approaches to wound management, Clin. Microbio. Rev. 14 (2) (2001) 244–269, https://doi.org/10.1128/CMR.14.2.244–269.2001. [3] K. Vowden, P. Vowden, Wound dressings: principles and practice, Surgery (Oxford) 32 (2014) 462–467. [4] P.L. Bigliardi, S.A.L. Alsagoff, H.Y. El-Kafrawi, J.K. Pyon, C.T. Cheuk Wa, M.V. Anthony, Povidone iodine in wound healing: a review of current concepts and practices, Int. J. Surgery. 44 (2017) 260–268, https://doi.org/10.1016/j.ijsu.2017.06.073. [5] N. Mayet, Y. Choonara, P. Kumar, L. Tomar, C. Tyagi, D. Toit, V. Pillay, A comprehensive review of advanced biopolymeric wound healing systems, J. Pharm. Sci. (2014) 2211–2230, https://doi.org/10.1002/jps.24068. [6] T. Phaechamud, P. Issarayungyuen, W. Pichayakorn, Gentamicin sulfate-loaded porous natural rubber films for wound dressing, Int. J. Biol. Macromol. 85 (2016) 634–644, https://doi.org/10.1016/j.ijbiomac.2016.01.040. [7] S. Anjum, A. Arora, M.S. Alam, A.B. Gupta, Development of antimicrobial and scar preventive chitosan hydrogel wound dressings, Int. J. Pharm. 508 (2016) 92–101. [8] B. Singh, L. Varshney, S. Francis, Rajneesh, Designing tragacanth gum based sterile hydrogel by radiation method for use in drug delivery and wound dressing applications, Int. J. Biol. Macromol. 88 (2016) 586–602, https://doi.org/10.1016/j.ijbiomac. 2016.03.051. [9] R. Fu, C. Li, C. Yu, H. Xie, S. Shi, Z. Li, Q. Wang, L. Lu, A novel electrospun membrane based on moxifloxacin hydrochloride/poly(vinyl alcohol)/sodium alginate for antibacterial wound dressings in practical application, Drug Deliv 23 (2016) 828–839, https://doi.org/10.3109/10717544.2014.918676. [10] M. Tummalapalli, M. Berthet, B. Verrier, B.L. Deopura, M.S. Alam, B. Gupta, Composite wound dressings of pectin and gelatin with aloe vera and curcumin as bioactive agents, Int. J. Biol. Macromol. 82 (2016) 104–113, https://doi.org/10.1016/j.ijbiomac. 2015.10.087. [11] M.H. Asfour, H. Elmotasem, D.M. Mostafa, A.A. Salama, Chitosan based Pickering emulsion as a promising approach for topical application of rutin in a solubilized form intended for wound healing: in vitro and in vivo study, Int. J. Pharm. 534 (2017) 325–338, https://doi.org/10.1016/j.ijpharm.2017.10.044. [12] W.Y. Ng, A. Migotto, T.S. Ferreira, L.B. Lopes, Monoolein-alginate beads as a platform to promote adenosine cutaneous localization and wound healing, Int. J. Biol. Macromol. 102 (2017) 1104–1111, https://doi.org/10.1016/j. ijbiomac.2017.04.094. [13] E.E. Ozseker, A. Akkaya, Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads, Carbohydr. Polym. 20 (2016) 441–451, https://doi.org/10.1016/j.carbpol.2016.05.073. [14] A. Osmokrovic, I. Jancic, J. Vunduk, P. Petrovic, M. Milenkovic, B. Obradovic, Achieving high antimicrobial activity: composite alginate hydrogel beads releasing activated charcoal with an immobilized active agent, Carbohydr. Polym. 15 (2018) 279–288, https://doi.org/10.1016/j.carbpol.2018.05.045. [15] J. Qu, X. Zhao, Y. Liang, T. Zhang, P. Ma, B. Guo, Antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing, Biomaterials 183 (2018) 185–199, https://doi.org/ 10.1016/j.biomaterials.2018.08.044. [16] X. Zhao, H. Wu, B. Guo, R. Dong, Y. Qiu, P. Ma, Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing, Biomaterials 122 (2017) 34–47, https://doi.org/10.1016/j.biomaterials.2017.01.011. [17] J. Li, C. Jiang, X. Lang, M. Kong, X. Cheng, Y. Liu, C. Feng, X. Chen, Multilayer sodium alginate beads with porous core containing chitosan based nanoparticles for oral delivery of anticancer drug, Int. J. Biol. Macromol. 85 (2016) 1–8, https://doi.org/10. 1016/j.ijbiomac.2015.12.064. [18] T. Agarwal, S.N. Narayana, K. Pal, K. Pramanik, S. Giri, I. Banerjee, Calcium alginatecarboxymethyl cellulose beads for colon-targeted drug delivery, Int. J. Biol. Macromol. 75 (2015) 409–417, https://doi.org/10.1016/j.ijbiomac.2014.12.052. [19] D. Bansal, A. Gulbake, J. Tiwari, S.K. Jain, Development of liposomes entrapped in alginate beads for the treatment of colorectal cancer, Int. J. Biol. Macromol. 82 (2016) 687–695, https://doi.org/10.1016/j.ijbiomac.2015.09.052. [20] A. Pandit, G. Divase, T. Chavan, K.R. Khandelwal, Oral lipid based multiparticulate pastilles: design and effect of pore former, J. Pharm. Investig. (2015) 23–33, https://doi.org/10.1007/s40005-014-0141-4. [21] X. Zhao, P. Li, B. Guo, P. Ma, Antibacterial and conductive injectable hydrogels based on quaternized chitosan-graft-polyaniline/oxidized dextran for tissue engineering, Acta Biomater. 26 (2015) 236–248, https://doi.org/10.1016/j. actbio.2015.08.006. [22] N. Sanchez-Ballester, I. Soulairol, B. Bataille, T. Sharkawi, Flexible heteroionic calcium-magnesium alginate beads for controlled drug release, Carbohydr. Polym. 1 (2019) 224–229, https://doi.org/10.1016/j.carbpol.2018.11.096. [23] A. Pandit, S. Patel, V. Bhanushali, V. Kulkarni, V. Kakad, Nebivolol loaded microsponge gel for healing of diabetic wound, AAPS PharmSciTech 18 (2017) 846–854, https://doi.org/10.1208/s12249-016-0574-3. [24] N. Ninan, M. Muthiah, N. Kalarikkal In-KyuPark, A. Elain, T. Wong, S. Thomas, Y. Grohens, Pectin/carboxymethyl cellulose/microfibrillated cellulose composite scaffolds for tissue engineering, Carbohydr. Polym. 15 (2013) 877–885, https://doi. org/10.1016/j.carbpol.2013.06.067. [25] M. Buriuli, W. Kumari, D. Verma, Evaluation of hemostatic effect of polyelectrolyte complex-based dressings, J. Biomater. Appl. 32 (2017) 638–647, https://doi.org/ 10.1177/0885328217735956.
A.P. Pandit et al. / International Journal of Biological Macromolecules 140 (2019) 1106–1115 [26] N. Devi, J. Dutta, Preparation and characterization of chitosan-bentonite nanocomposite films for wound healing application, Int. J. Biol. Macromol. 104 (2017) 1897–1904, https://doi.org/10.1016/j.ijbiomac.2017.02.080. [27] L. Bennison, C. Miller, R. Summers, A. Minnis, G. Sussman, W. McGuiness, The pH of wounds during healing and infection: a descriptive literature review, Wound Practice and Research vol. 25 (2017) 63–69. [28] K. Kathe, H. Kathpalia, Film forming systems for topical and transdermal drug delivery, Asian J. Pharm. Sci. 2 (2017) 487–497, https://doi.org/10.1016/j.ajps.2017.07. 004. [29] J.S. Boateng, H.V. Pawar, J. Tetteh, Polyox and carrageenan based composite film dressing containing anti-microbial and anti-inflammatory drugs for effective wound healing, Int. J. Pharm. 441 (2013) 181–191, https://doi.org/10.1016/j. ijpharm.2012.11.045. [30] M.D. Romic, M.S. Klaric, J. Lovric, I. Pepic, B.C. Cizmek, J.F. Grcic, A. Hafner, Melatoninloaded chitosan/Pluronic® F127 microspheres as in situ forming hydrogel: an innovative antimicrobial wound dressing, Eur. J. Pharm. Biopharm. 107 (2016) 67–79, https://doi.org/10.1016/j.ejpb.2016.06.013. [31] A. Shrivastav, A.K. Mishra, S.S. Ali, A. Ahmad, M.F. Abuzinadah, N.A. Khan, In vivo models for assessment of wound healing potential: a systematic review, Wound Med 20 (2018) 43–53.
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[32] S. Murthy, M.K. Gautam, Shalini Goel, V. Purohit, H. Sharma, R.K. Goel, Evaluation of in vivo wound healing activity of Bacopa monniera on different wound model in rats, BioMed Res. Int 972028 (2013) 9, https://doi.org/10.1155/2013/972028. [33] S. Mahapatra, S. Mohanta, A. Nayak, Preliminary investigation of the angiogenic potential of Ziziphus oenoplia root ethanolic extract using the chorioallantoic membrane model, ScienceAsia 37 (2011) 72–74, https://doi.org/10.2306/ scienceasia1513-1874.2011.37.072. [34] N. Pettinelli, S. Rodríguez-Llamazares, V. Abella, L. Barral, R. Bouza, Y. Farrag, F. Lago, Entrapment of chitosan, pectin or κ-carrageenan within methacrylate based hydrogels: effect on swelling and mechanical properties, Mater. Sci. Eng. C Mater. Biol. Appl. 96 (2019) 583–590, https://doi.org/10.1016/j.msec.2018.11.071. [35] F. Shamekhi, E. Tamjid, K. Khajeh, Development of chitosan coated calcium-alginate nanocapsules for oral delivery of liraglutide to diabetic patients, Int. J. Biol. Macromol. 120 (2018) 460–467, https://doi.org/10.1016/j.ijbiomac.2018.08.078. [36] E. Salmerón-González, E. García-Vilariño, A. Ruiz-Cases, A. Sánchez-García, J. GarcíaSánchez, Absorption capacity of wound dressings: a comparative experimental study, Plast. Surg. Nurs. 38 (2018) 73–75, https://doi.org/10.1097/PSN. 0000000000000218.
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Spongy wound dressing of pectin/carboxymethyl tamarind seed polysaccharide loaded with moxifloxacin beads for effective wound heal Ashlesha P. Pandit a,⁎, Kanchan R. Koyate a, Ashwini S. Kedar a, Vaishali M. Mute b a b
Department of Pharmaceutics, JSPM Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India Department of Pharmacology, JSPM Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 25 April 2019 Received in revised form 22 August 2019 Accepted 23 August 2019 Available online 27 August 2019
a b s t r a c t An attempt was made to formulate moxifloxacin loaded alginate beads incorporated into spongy wound dressing to heal chronic wounds as well as to reduce frequency of painful dressing change. Moxifloxacin loaded beads (sodium alginate:pectin, 1:1) were prepared by ionic gelation method, with entrapment efficiency 94.52%, crushing strength 25.30 N and drug release 90.52%. Beads were further incorporated into wound dressing, made of pectin and carboxymethyl tamarind seed polysaccharide (CMTSP). Spongy wound dressing was obtained by freeze drying technology, which showed good folding endurance, high wound fluid absorption and good crushing strength. Drug release was found to be 85.09%. Dressing made of CMTSP:pectin (1.5:2) showed good water vapour transmission and antibacterial activity. Porous nature of dressing absorbed exudates of wound. Excision wound model in rats revealed wound healing within 17 days: groups I (control), II (moxifloxacin beads loaded wound dressing), III (moxifloxacin beads), IV (pectin film) and V (sodium alginate film) showed 65.28, 99.09, 86.90, 66.84 and 64.30% wound closure, respectively. To conclude, moxifloxacin beads loaded spongy wound dressing has good healing and wound closing potential compared to pectin film and moxifloxacin beads. Thus, the formulation is novel for biomedical application which reduced the frequency of painful dressing change. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Wound healing is a major health care challenge. With an unparalleled increase in incidence of chronic wounds at accidents, traumatic wounds and complicated post-surgical scenario, it is highly desirable to design a high performance antibacterial wound-care system [1]. A wound can be defined as an injury or disruption to the skin and can extend to subcutaneous tissue, muscles, tendons, nerves, vessels, and even to the bone [2]. Based on the nature and repair process wounds can be classified as chronic and acute wound. However, chronic wounds are more prone to contamination and usually involve significant tissue loss that can affect vital structures such as bones, joints, and nerves [3]. To overcome this problem, wound dressings are used to protect the wound from external environment and contamination. Topical bioactive agents like solutions, creams, and ointments are also used to cure wound, but are less effective due to rapid fluid absorption, thus become mobile losing rheological characteristics [4]. Therefore, solid wound dressings are preferred in the case of exudate wounds, due to better exudate management and prolonged residence at the wound site than other formulations. Currently, advanced dressings of bioactive agents ⁎ Corresponding author at: JSPM Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India. E-mail address: [email protected] (A.P. Pandit).
https://doi.org/10.1016/j.ijbiomac.2019.08.202 0141-8130/© 2019 Elsevier B.V. All rights reserved.
are used to get faster wound healing [5]. However, porous wound dressings are treatment of choice as these dressings absorb wound fluid exudates and also allow water vapour transmission [6]. Moreover, wound dressing efficiency is enhanced by loading beads into it. These beads are loaded with antibacterial drug to sustain release of drug and are prepared by ionic gelation method. Antibiotics are of choice to treat wound infection [7]. However, one of them is moxifloxacin, for prevention and elimination of microorganisms at wound sites. Moxifloxacin (MX) is a fourth-generation fluoroquinolone antibiotic, with similar in vitro activity as that of ciprofloxacin and ofloxacin against Gram-negative bacteria, but enhanced activity against Gram-positive bacteria [8]. Moxifloxacin is beneficial for topical treatment of infected wound as it accelerates wound repair process [9]. Pectin is a natural prophylactic substance used for wound healing which acts against poisoning with toxic cations. Pectin has styptic and curing effects which are well documented in healing ointments [10]. However, due to its poor intrinsic mechanical properties, it is used typically in conjunction with other polymers. Extensive literature revealed that researchers developed a ternary nano dressing consisted of titanium dioxide nano particle loaded chitosan-pectin [11]. Beneficial effects of beads in wound care has been studied thoroughly as follows: alginate beads containing the polar lipid monoolein to manage wet wounds by providing improved uptake of excess exudate while releasing adenosine locally for promotion of healing [12]; calcium-alginate beads to immobilize tetracycline
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and improve an antibacterial biomaterial for open wound [13]; new composites based on Ca-alginate hydrogels released activated charcoal (AC) particles with adsorbed povidone iodine (PVP-I) as a model antimicrobial substance in a physiological-like environment [14]. Recently, newer approach is the use of antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility of wound dressing for joints skin wound healing [15] and novel antibacterial anti-oxidant electro-active injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing [16]. Currently, wound dressings are of choice to heal wound. However, changing of dressing is uncomfortable to the patients. Time required to heal wound reduces their quality of life. Dressing changes also represent a major financial and logistical burden to patients. Keeping in view the above problems, dressing change frequency was planned to reduce by using biomaterials such as pectin, carboxymethyl cellulose and alginate which themselves have wound healing potential. Thus, the aim was to formulate porous wound dressing embedded with moxifloxacin-alginate beads. Dressing was made of combination of pectin and carboxymethyl tamarind seed polysaccharide for better wound care. Application of this novel dressing was planned to reduce the frequency as well as pain which occurred during dressing change, each time. Beads loaded dressing allowed slow release of MX at the wound area. Advanced treatment modality of freeze dried technology thought to make the dressing spongy which helped to absorb exudates of wound and permeated water vapour transmission, required for fast wound heal. 2. Materials and methods 2.1. Materials MX was received as gift sample from Macleods Pharmaceuticals, India. Pectin, sodium alginate and nutrient agar were procured from Hi-media, Mumbai, India. Carboxymethyl tamarind seed polysaccharide (CMTSP) was kindly gifted by Alfa Exim, India. 2.2. Formulation of MX beads Beads were prepared by ionic gelation method using sodium alginate and pectin [17]. Sodium alginate: pectin ratios 1:1 (B1), 1:1.5 (B2), and 1:2 (B3) were added in distilled water (20 ml) and stirred for 15 min. MX (500 mg) was added to the above solution and again stirred. Polymer solution thus obtained was added drop by drop using syringe to the calcium chloride (20%) solution to get spherical beads. Beads were filtered and washed three times with distilled water [18]. 2.3. Entrapment efficiency of beads Entrapment efficiency of MX-loaded beads (MX-B) is the total amount of drug present in the product. Beads were washed with distilled water and were analyzed to calculate the amount of free drug. Entrapment efficiency was calculated using Eq. (1) [19]: Entrapment Efficiency ð%Þ ¼ Total drug added−Free drug=Total drug added 100
ð1Þ
2.4. Crushing strength of beads Crushing strength is the maximal force corresponding to the peak of the force-time plot. Crushing strength of the beads was determined using a CT3 Texture Analyzer (Brookfield Engineering Laboratories, Inc., USA), operating at 0.05 N load cell. Thereafter, force (N) vs time (s) plot was recorded using texture analyzer software. In short, a spherical bead was placed on lower flat plate. The upper punch of 25.4 mm
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diameter and 35 mm length, made of clear acrylic material, was moved down, slowly at a constant rate of 0.3 mm/s [20] (Texture Pro CT). 2.5. Antibacterial activity Antibacterial activity of MX (MX 500 μg dissolved in 0.5 ml DMSO) as well as MX-loaded beads (equivalent to 500 μg MX) against causative microorganisms of wound infection S. aureus [21] and P. aeruginosa was evaluated using agar diffusion method [8]. Herein, sterile nutrient agar media was poured into sterilized plate under a laminar airflow unit. Thereafter, diluted suspension of microbe (standardized concentration of 108 cells/ml) was spread onto filled agar plate and was kept for solidification. Next, cups were punched on agar plate and filled with solution or beads, separately. Petri plates were then incubated at 37 °C for 24 h (n = 3). Lastly, the zone of inhibition was measured and reported as mean ± S.D. [22]. 2.6. In-vitro drug release of beads In vitro release of MX-B was performed using modified USP dissolution test apparatus (Basket type, TDT 08, Electro lab, India). Initially, beakers (250 ml) were placed in dissolution flasks of the dissolution tester and were filled with phosphate buffer solution pH 7.4 (100 ml). An accurately measured amount of beads, equivalent to 500 μg MX, were filled in baskets and suspended in beakers containing dissolution media. Thereafter, the apparatus was run at 150 rpm at 37 ± 0.5 °C. Further, aliquots of samples (5 ml) were collected till 8 h and analyzed by UV spectrophotometer at λmax 292 nm. Sink condition was maintained in dissolution medium by replacing aliquots each time with same volume at 37 ± 0.5 °C [6,23]. 2.7. Scanning electron microscopy Morphology and surface topography of bead was studied using SEM. Prepared beads were coated with gold–palladium under an argon atmosphere at room temperature and then the surface morphology of the beads was studied by scanning electron microscopy at 50× and 500× magnification (JEOL Oxford). 2.8. Formulation of beads loaded wound dressing Porous wound dressing of MX-loaded beads (MX-BWD) was prepared by freeze drying method [24]. Briefly, aqueous polymer blends of CMTSP (1.0 and 1.5 g): pectin (1.0 and 2.0 g) and glycerin (1 ml) were homogenized for 15 min (Table 1). Polymeric solution (2 ml), thus formed, was poured into glass mold (dimensions: 3 × 3 cm) and weighed amount of beads (equivalent to single dose of 500 μg) were uniformly spread into the mold. Further, beads were covered by adding remaining 2 ml of polymer solution. The resulting solution was then subjected to freeze drying technology (Martin Christ, Germany, Alpha 1–2 LD Plus) at −42.5 °C for 48 h to get porous mass [25]. The obtained spongy wound dressing (F1 to F4) was stored until further use. 2.9. Characterization of beads loaded wound dressing Wound dressing was characterized for thickness using micrometer screw gauge. The pH of dressing was determined by immersing MXBWD in normal saline solution till it reached equilibrium. Dressings were then removed from the solution and pH of the solution was determined using digital pH meter. All tests were performed in triplicate and average values were recorded [26]. Next, the folding endurance of dressing was measured manually. It is the number of times the patch is folded at the same place to break the patch. It provides an indication of brittleness [27]. Tensile strength was studied using texture analyzer (CT3 Texture Analyzer, Brookfield Engineering Labs. Inc., USA) with
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Table 1 Formulation of beads loaded spongy film dressing. Batch
MX (μg)
CMTSP (g)
Pectin (g)
Thickness (mm)
pH
Folding endurance
Tensile strength (N)
Wound exudate absorptive capacity (%)
Drug release (%)
F1 F2 F3 F4
500 500 500 500
1.0 1.0 1.5 1.5
1. 0 2.0 1.0 2.0
0.41 ± 0.012 0.47 ± 0.01 0.44 ± 0.012 0.5 ± 0.015
5.87 5.42 6.16 5.26
148 ± 4 164 ± 12 143 ± 7 161 ± 4
8.87 ± 1.1 11.17 ± 1.2 9.16 ± 0.77 15.88 ± 0.65
683.99 ± 3.28 606.89 ± 4.37 578.63 ± 4.72 552.07 ± 2.89
85.21 ± 2.11 80.83 ± 0.89 85.62 ± 1.09 85.09 ± 2.03
TexturePro CT software. Dressing (2 × 5 cm) was cut and fixed between two clamps of texture analyzer with a load of 50 N. The attached strip was then pulled at the rate of 10 mm/min. Tensile strength at break of dressing was calculated by following Eq. (2): Tensile strength ¼ ðbreaking forceÞ= cross−sectional area N=m2
ð2Þ
2.10. In-vitro drug release of MX-BWD Wound dressings were immersed in beakers (capacity 500 ml) containing 100 ml phosphate buffer solution (pH 7.4) at USP dissolution test apparatus Type II (paddle type) [18], at 37 ± 0.5 °C at 50 rpm. At predetermined time, aliquot of 5 ml was withdrawn at an interval of 1 h till 10 h and replaced with same volume of fresh phosphate buffer solution. Then, the volume of samples were diluted up to 10 ml and analyzed by UV spectroscopy at 292 nm [8]. 2.11. Wound exudates absorptive capacity Fluid absorption capacity of wound dressings was measured by using simulated wound fluid (SWF). SWF was prepared by dissolving 0.02 M calcium chloride, 0.4 M sodium chloride, 0.08 M methylamine, and 2% w/v of bovine serum albumin in distilled water (100 ml) at pH 7.4. The previously weighed dressing (3× 3 cm) was dipped in SWF. After, fixed time interval, swollen dressing was wiped with cotton to remove excess SWF on the surface, and re-weighed. This was repeated until a constant weight was obtained. Thereafter, change in the weight of film was noted [28,29]. Wound exudates absorptive capacity (%) was calculated using Eq. (3): Wound exudates absorptive capacity ð%Þ ¼ ðWs−WdÞ=Wd 100 ð3Þ where Ws and Wd are weight of the swollen and dry film, respectively. 2.12. Water vapour transmission Water vapour transmission rate was determined by fixing MX-BWD (20 mm diameter) to Franz diffusion cell containing distilled water (20 ml). An open cell was used as a control. A desiccator, was kept in a hot air oven at 35 °C. Diffusion cell was placed in desiccator, which was further re-weighed at periodic intervals (1, 3, 5, 7, 9, 12, 24 h). Here, weight loss corresponds to evaporated water content. Thereafter, water vapour transmission rate was calculated using following Eq. (4) [26,30]: Water vapour transmission ¼ ml 24=tS
ð4Þ
where ml is mass loss during the time t (measured in h) and S is the area of cell mouth.
2.14. Degradability study of dressing Wound dressing was subjected to degradation study based on the earlier reported method [21]. Briefly, dressings were weighed (900 mg, 2 × 2 cm) and immersed in 20 mL SWF medium (pH 7.4) with continuous shaking at 100 rpm at 37 °C. Initial weight of the dressing was noted as Wi. Each set of dressings were removed at time interval of 7, 14, 21 days from the medium, washed with deionized water to clean ions adsorbed on surface and dried in an oven at 50 °C for 48 h and then reweighed (Wt). The degradation of dressing was calculated as follows: Degradation ð%Þ ¼ ½ðWi−WtÞ=Wi 100
ð5Þ
2.15. In-vivo wound healing study Healthy male Wistar albino rats (60 days old), weighing between 150 and 200 g were acclimatized to the laboratory environment for a period of 3 days. Animals were housed at temperature 22 ± 1 °C and humidity 35 ± 5% to 65 ± 5% RH in clean sterile polycarbonate cages containing autoclaved paddy husk, with free access to water and food, and kept under 12/12 h light/dark cycles. All the experimental procedures and protocol were approved by the Institutional Animal Ethics Committee, India, constituted under Committee for Purpose of Control and Supervision of Experiments on Animals (IAEC-16-011). Animals were divided into five groups containing 3 animals in each group. Group I: control (CG); Group II: moxifloxacin beads loaded in spongy wound dressing (MX-BWD); Group III: moxifloxacin beads (MX-B); Group IV: pectin film (PF); and Group V: sodium alginate film (SAF). 2.16. Excision wound model Animals were anaesthetized with anaesthetic ether by open mask method and shaved on both sides of back with electric clipper. Thereafter, excision wound was inflicted on the dorsal thoracic region 1–1.5 cm away from the vertebral column and 5 cm away from ear of each animal. A full thickness excision wound of circular area 100 mm2 and 2 mm in depth was created along the markings. The entire wound was left open. Animals were closely observed for any infection and those which showed signs of infection were separated and excluded from the study. Spongy dressing was applied on the wound once, on day 0 to cover the wound [31,32]. Wound areas were measured planimetrically on days 1, 7, 14, and 17 and percent wound closure was calculated using Eq. (6): Wound closure ð%Þ ¼ Wound area on 0 day−Wound area on nth day Wound area on 0 day 100
ð6Þ
where, n is the number of days (1, 7, 14 and 17). 2.17. Histological study
2.13. Antibacterial activity Antibacterial activities of (a) pectin and CMTSP against P. aeruginosa, (b) MX-BWD against P. aeruginosa, (c) pectin and CMTSP against S. aureus, (d) MX- BWD against S. aureus were performed as discussed previously, in Section 2.5. The zone of inhibition was measured in mm [13].
Rats were sacrificed on day 17 after complete wound heal. Next, wounded area of skin, containing dermis and hypodermis, was isolated and carefully trimmed with cutter. It was then fixed in 10% neutral formalin solution. After paraffin embedding, 3 to 4 μm sections were stained with hematoxylin and eosin (H and E) for the study of tissue appearance. All the slides were observed under light microscopy [11].
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2.18. Statistical analysis
3.1. Entrapment efficiency
Experimental results were expressed as the mean standard error using software GraphPad Prism (Version 5, GraphPad Software Inc., La Jolla, CA). The results were analyzed statistically using two-way analysis of variance (ANOVA) followed by Bonferroni post-tests, where P b 0.001 was considered as statistically significant [32].
The amount of increased pectin concentration (1.0 to 2.0 g) resulted in decreased entrapment efficiency of drug (94.52 ± 1.5, 93.56 ± 1.3 and 89.83 ± 2.1%). Calcium ions induce chain–chain associations that formed junction zones (called egg-box-junctions) which was responsible for gel formation by both sodium alginate and pectin. The chemical interactions occur between calcium ions and guluronate and galacturonate blocks present in sodium alginate and pectin, respectively. Thus, increase in pectin concentration, decreased an entrapment efficiency. The obtained result is in agreement with previous results [19].
2.19. Cell compatibility of wound dressing Cell compatibility of wound dressing was studied based on the mechanism of angiogenesis. Angiogenesis is a complex physiological process required for healing the wounds, which involves restoring blood flow to tissues after injury, has become a major focus of study for wound biologists. Angiogenic activity of wound dressing was studied using an in vitro chick chorioallantoic membrane (CAM) model [33]. The CAM is a vascular extra-embryonic membrane found in eggs of some amniotes, such as chick. It is formed on day 4 of incubation. Briefly, nine days old fertilized chick eggs were selected. A small window in the egg shells was opened, carefully. Then, a sterile wound dressing (2 × 2 cm) was positioned at the joint of two large blood vessels. The window was resealed with adhesive tape, cautiously and the eggs were incubated further at 37 ± 1 °C in a well-humidified chamber. Next, the tape was opened after 72 h to observe new blood vessel formation. The results were compared with the control eggs without the dressing.
2.20. Stability study Stability study of wound dressing was studied to obtain a stable product which assures safety and efficacy, till shelf life, at defined storage and package conditions. Accelerated stability study was performed according to ICH guidelines to assess the combined effect of excipients of beads and dressing on stability of the dressing [23]. Dressing was packed in amber color bottle, sealed, and kept at stability chamber (Thermolab, India) at 40 ± 2 °C/75 ± 5% RH. The samples were evaluated for pH, folding endurance, microbial growth, strength, entrapment efficiency and drug release after 7 and 15 days and 1, 3, and 6 months.
3. Results and discussion MX-B were formulated by using sodium alginate and pectin by ionic gelation method. Here, sodium alginate forms a viscous solution with water, while in contact with calcium chloride solution forms spherical beads.
3.2. Crushing strength Beads exhibited the maximal crushing strength at the highest amount of pectin (25.30, 25.52 and 26.36 N) (Fig. 1a). Crushing strength increased with increased amount of pectin. Pectin provides good strength to beads. Pectin, also known as pectic polysaccharides, is rich in galacturonic acid. It contains several distinct polysaccharides such as homogalacturonans which is linear chains of α-(1-4)-linked Dgalacturonic acid. Therefore, high amount of pectin provides good strength and rigidity to the beads [34]. 3.3. Antibacterial study Antimicrobial activity of MX and MX-B against S. aureus was 57.04 ± 0.65 mm and 56.02 ± 0.5 mm, respectively, and 59.4 ± 0.55 mm and 58.6 ± 0.43 mm, respectively against P. aeruginosa (Fig. 2a–d). The release of MX through beads in the media was delayed due to presence of pectin. Therefore, beads showed, comparatively, less zone of inhibition than MX. Thus, MX-B were potential candidate for effective wound dressing. 3.4. In vitro drug release study of MX-B Increase in concentration of pectin decreased the drug release from the beads MX-B. Herein, formulation B1 (1:1), B2 (1:1.5) and B3 (1:2) with increased concentration of pectin showed 23.40 ± 1.3, 22.41 ± 2.1 and 20.81 ± 1.8%, respectively, drug release in first 2 h; 47.67 ± 1.5, 42.48 ± 2.4 and 41.18 ± 2.2% release within 4 h; 69.39 ± 1.7, 68.10 ± 2.4 and 65.39 ± 1.1% release within 6 h; followed by 90.54 ± 1.6, 88.24 ± 1.5 and 87.68 ± 2.7% release, respectively in further 8 h (Fig. 3). Beads B1 and B2 at lower concentration of pectin released the drug rapidly than B3. However, presence of more amount of pectin at B3 forms thick layer on the surface of beads. Pectin gets swell in contact with dissolution media, thus delayed the release of MX than B1 and B2 due to slow dissolution of drug [7,35]. Here, all batches exhibited
Fig. 1. (a) Crushing strength of beads; tensile strength of (b) F4.
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Fig. 2. Antibacterial activity of (a) moxifloxacin against P. aeruginosa, (b) moxifloxacin beads against P. aeruginosa, (c) moxifloxacin against S. aureus, (d) moxifloxacin beads S. aureus.
linear release. However, at high amount of pectin, the behavior of release of MX though beads could be non-linear.
helped to enhance the wound exudates absorptive capacity [24]. Also, the wound exudate enters in spongy dressing and provides medium to release drug from beads.
3.5. Selection of batch of beads 3.8. Characterization of MX-BWD Beads B1 (1:1), B2 (1:1.5) and B3 (1:2) showed entrapment efficiency of 94.52 ± 1.5, 93.56 ± 1.3 and 89.83 ± 2.1%, respectively, within 8 h. Increase in pectin concentration decreased the entrapment efficiency. The crushing strength of beads was not much affected for B1 (25.30 N) and B2 (25.52 N), although, found to be increased in B3 (26.13 N). The release of MX through B1, B2 and B3 was 90.52, 88.47 and 88.23%, respectively, within 8 h. The effect of pectin in sodium alginate: pectin beads exhibited results as follows: highest amount of drug was entrapped in beads B1 with drug release within 8 h with good crushing strength, while fewer amount of drug was entrapped in B2 and B3. Therefore, formulation B1 was selected for further studies. 3.6. Scanning electron microscopy (SEM) SEM of the beads revealed spherical structure (Fig. 4a) with rough surface. Wherein, spherical nature of beads was required to load the beads in the dressing. Similar result was obtained in earlier study for sodium alginate beads which exhibited rugged surface [13]. 3.7. Formulation of MX-BWD Wound dressing is an advanced technology, used for treatment of acute as well as chronic wounds. Herein, MX-B were prepared by ionic gelation method using sodium alginate and pectin. Beads were loaded into dressing base consisted of pectin and CMTSP (Fig. 4b). Next, dressing was made spongy by freeze drying method (Fig. 4c). Spongy nature of dressing imparted flexibility in movements to the patient as well as
Dressing thickness measurement is often vitally important for absorbing the exudate of wound in the spongy mass. Thickness of MXBWD varied in the range of 0.41 ± 0.012 to 0.5 ± 0.015 mm. F1 dressing was less thick, while F4 showed highest thickness amongst all the formulations. Thick wound dressing is important parameter to provide sufficient strength as well as impart spongy nature [26]. High folding endurance of dressing is the indication of flexibility and elasticity that allows the free movements of body parts after application [26]. F2 exhibited highest folding endurance at 163 ± 12, while F3 showed lowest at 143 ± 7. High concentration of pectin showed more flexibility and therefore, depicted the highest folding endurance [28]. Tensile strength testing provides an indication of the strength and elasticity of the dressing. It is a versatile tool for the mechanical characterization of pharmaceutical film dressings. F4 dressing exhibited the highest tensile strength at 15.88 ± 0.65 N (Fig. 1b). The addition of higher amount of pectin increased the tensile strength of the dressing. F1 shows the tensile strength of 8.87 ± 1.1 N. High tensile strength is always favored that prevents the breaking of dressing during application, storage and transportation [35]. Wound dressing shows the pH in the range between 5.26 and 6.16. These values are in close agreement with the dressing pH values (5.25–7.90) reported earlier [26]. Dressing pH is a vital parameter for wound dressing, which favors to regulate infection at wound surface as well as accelerates the formation of fibroblast proliferation. The pH of normal human skin ranges between 4.0 and 6.8, therefore, pH of wound dressing should be slightly acidic at wound surface, which thus helps to accelerate the wound heal. An alkaline wound
Fig. 3. Drug release through beads.
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Fig. 4. Scanning electron microscopy of moxifloxacin beads (a) 50× magnification, (b) distribution of beads in wound dressing (c) porous wound dressing.
pH is associated with reduced wound healing process. Open wounds characteristically have a neutral to alkaline pH that ranges 6.5 to 8.5, while chronic wounds 7.2 to 8.9. An alkaline wound environment impairs the healing and immunological response by promoting bacterial growth increasing proteolytic activity, inhibiting fibroblasts and decreasing oxygen supply. 3.9. In-vitro drug release Wound dressing F2 and F4 with high amount of pectin resulted in 37.27 and 41.45% drug release, respectively, within first 5 h, followed by 80.83 and 85.09% drug, respectively within 10 h. Formulation F1 and F3 showed 42.37 and 39.16% within first 5 h, followed by 85.21 and 85.62% drug release, respectively within 10 h as shown in Fig. 5. Thus, it was observed that increased concentration level of CMTSP delayed the drug release from the wound dressing and resulted in slow release of drug from the wound dressing compared to other formulations [24]. However, drug release behaves differently at high levels of CMTSP. The release of MX through dressing crosses the swollen dressing due to CMTSP as well as embedded beads in it. This resulted in comparatively delayed MX release through dressing than beads. 3.10. Wound exudates absorptive capacity Wound exudates absorption capacity of dressing was due to two processes: water absorbed by materials and water being retained in
the porous structure. Dressings absorbed 483.99 ± 0.28 to 606.89 ± 0.37% of SWF within 60 min. Fast absorption of exudates into dressing is vital for fast wound healing, because presence of high amount of exudate slows down cell proliferation. Moreover, wound exudate retained at porous wound dressing provides media to release drug from beads. Wound exudates absorption capacity of dressing was found to be directly proportional amount of pectin due to its water retention capacity. High wound exudates absorptive capacity indicated structural integrity of the dressing [35,36].
3.11. Water vapour transmission Moisture regulation of wound is an important aspect and results in getting wound conditions more amenable to healing. The dried wounds can delay the healing process, while accumulation of exudates influences the occurrence of the infection. Therefore, capacity to exchange the moisture through the dressing at optimum level is required to fast heal the wound. Evaporative water loss for normal skin is 204 ± 12 g/m2/day; while for injured skin range from 279 ± 26 g/m2/day to 5138 ± 202 g/m2/day for a granulating wound. Herein, an adequate level of moisture, is required to avoid risk of wound dehydration as well as buildup of exudates on the wound, water vapour transmission rate from injured wound should be in the range 2000–2500 g/m2/day [30]. The prepared MX-BWD (F4) showed weight loss and water vapour transmission rate of 4.15 and 31.71 ± 18 g/m2/day, respectively after 1 h; 7.82 g and 59.77 ± 19 g/m2/day, respectively after 3 h; 12.14 g
Fig. 5. Drug release through wound dressing.
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Fig. 6. Antibacterial activity of (a) pectin and CMTSP against P. aeruginosa, (b) wound dressing against P. aeruginosa, (c) pectin and CMTSP against S. aureus, (d) wound dressing against S. aureus.
and 92.78 ± 27 g/m2/day, respectively after 5 h; 15.73 and 120.22 ± 25 g/m2/day, respectively after 7 h; 17.61 g and 134.59 ± 46 g/m2/day, respectively after 9 h; 26.98 g and 201.63 ± 49 g/m2/day, respectively after 12 h; 42.24 g and 322.85 ± 51 g/m2/day after 24 h, thus, close to the adequate range of the ideal wound dressing. Such water vapour transmission rate can strengthen the cellular re-epithelialization. Dressing F4 showed good water vapour transmission rate because of the highest concentration of both polymers [27]. Porous structure of wound dressing allowed water vapour transmission to a great extent which favors fast wound heal [7]. Decreased oxygen tension lowers the pH which affects angiogenesis, immunological activity and collagen formation. 3.12. Antibacterial activity CMTSP did not show zone of inhibition against both P. aeruginosa and S. aureus, while wound dressing exhibited nearly same zone as that of beads. Zone of inhibition of pectin and wound dressing against P. aeruginosa was 13.2 ± 0.3 and 57.8 ± 0.32, respectively (Fig. 6a and b). While, zone of inhibition of pectin, CMTSP and wound dressing against S. aureus was 11.8 ± 0.2, 12.2 ± 0.4 and 55.9 ± 0.45 mm, respectively (Fig. 6c and d). MX-BWD was effective against S. aureus and P. aeruginosa (zone of inhibition 55.9 ± 0.45 mm and 57.8 ± 0.32 mm, respectively). Wound dressing showed same zone of inhibition against both the Gram positive and Gram negative strains of bacteria as that of MX-B. Therefore, wound dressing was considered to have good antibacterial activity. 3.13. Degradability study of dressing Wound dressings degraded 8–11% within 7 days, 18–23% within 14 days and 28–38% within 21 days. Pectin has potential to bind to CMTSP and protect the dressing from degradation. Also, higher swelling and porosity account for increased biodegradation. MX was released within 10 h while porous dressing remained at the wound area till degradation. Thus, the above results accurately forecasted degradation profile of the dressing. The degradable characteristic would contribute to the reduced frequency of wound dressing change.
3.14. In-vivo study (excision wound model) Wound healing activity of the wound dressing was studied in the rat using excision wound model. Wound closure (%) of Group I: control (CG); Group II: F4 (MX-BWD); Group III: B1 (MX-B); Group IV: (PF); Group V: (SAF) on days 1, 7, 14 and 17 are as shown in Table 2 and Fig. 7. Comparison between Group I (CG) vs Group II (MX-BWD) of excision wound model using Two-way ANOVA is as follows: On day 1, MX-BWD showed 11.81% and CG showed 6.61% wound closure. It was found statistically significant with P˂0.001. On day 7, MX-BWD showed 42.20% and CG showed 16.44% wound closure. CG and MXBWD showed 25.76% difference in wound closure. It was found statistically significant (P ˂ 0.001). On day 14, MX-BWD showed 90.90% and CG showed 46.28% wound closure. It was found statistically significant (P ˂ 0.001). On day 17, MX-BWD and CG showed 99.09% and 65.28% wound closure, respectively. It was found statistically significant (P ˂ 0.001). PF showed 7.07% on day 1, 20.20% on day 7, 54.65% on day 14 and 66.84% on day 17 wound closure. SAF showed 6.59% on day 1, 27.17% on day 7, 47.82% on day 14 and 64.30% on day 17 wound closure. Group III (MX-B) showed 7.21% on day 1, 24.74% on day 7, 67.01% on day 14, and 86.59% on day 17 wound closure. MX-BWD treated rats, thus, showed faster healing within 17 days than MX-B loaded dressing, while PF and SAF showed very less healing of wound [1,31,32].
3.15. Histopathological study At the end of experiment, on day 17, rats of Group I (control) showed formation of scar (represented by arrow in Fig. 8a), severe inflammatory cell infiltration and fibroblast proliferation (represented by circle in Fig. 8a). Moreover, rats of Group II (MX-BWD) showed re-epithelialization (represented by arrow in Fig. 8b), and neovascularization (represented by small arrow in Fig. 8b) and regeneration of hair follicles (represented by circle in Fig. 8b). However, rats of Group III (MX-B) revealed formation of scar (represented by arrow in Fig. 8c), minimal inflammatory cell infiltration (represented by circle in Fig. 8c) and neovascularization (represented by small arrow in Fig. 8c) and healing under scar. Thereafter, rats of Group IV (PF) showed minimal formation of scar
Table 2 Rate of wound closure within seventeen days. Groups
Wound closure (%) on day 1 (Mean ± SD)
Wound closure (%) on day 7 (Mean ± SD)
Wound closure (%) on day 14 (Mean ± SD)
Wound closure (%) on day 17 (Mean ± SD)
Group I Group II Group III Group IV Group V
6.61 ± 0.72 11.81 ± 0.74 7.21 ± 0.58 7.07 ± 1.1 6.59 ± 0.79
16.44 ± 0.67 42.2 ± 1.1 24.74 ± 0.32 20.20 ± 0.75 27.17 ± 1.34
46.28 ± 0.55 90.90 ± 0.63 67.01 ± 0.175 54.65 ± 0.83 47.82 ± 0.72
65.28 ± 0.43 99.09 ± 0.76 86.59 ± 0.21 66.84 ± 0.64 64.30 ± 1.15
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Fig. 7. Comparative study of wound healing (excision wound model) in rats consist of five groups over a period of 17 days treatment. Values are ±SD of n = 5. Two-way ANOVA was used for statistical analysis. #P N 0.05. Group I: control; Group II: moxifloxacin beads loaded in wound dressing; Group III: moxifloxacin beads; Group IV: pectin film; Group V: sodium alginate film. Scale bar 5 mm.
tissue (represented by arrow in Fig. 8d). Rats of Group V (SAF) showed normal histological structure of wound (represented by Fig. 8e) [32]. Thus, the dressing MX-BWD exhibited rapid wound healing with closing of wound.
3.16. Cell compatibility of wound dressing A complex process of wound closing involves angiogenesis required for wound healing where its induction is beneficial in many clinical
Fig. 8. Histopathological study of wound tissue after 17 days, where (a) Group I (control), (b) Group II (moxifloxacin beads loaded wound dressing), (c) Group III (moxifloxacin beads), (d) Group IV (pectin film), (e) Group V (sodium alginate film). Scale bar 10 μm.
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References
Fig. 9. Cell compatibility of wound dressing using CAM model (a) control (b) dressing.
situations for achieving wound closure. Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels, formed in the earlier stage of vasculogenesis. During this process, proliferating capillaries bring oxygen and micronutrients to growing tissues and remove catabolic waste products. This leads to angiogenesis in wounds. In the chick CAM model, dressing showed an increase in density of blood capillaries on the treated membrane surface (Fig. 9a) compared to control group (Fig. 9b) of eggs. Thus, results indicated good angiogenic activity of dressing. Again, angiogenesis during wound repair serves the dual function of providing the nutrients demanded by the healing tissues and contributing structural repair through the formation of granulation tissue. 3.17. Stability study No significant change was observed at dressing F4 for pH (5.28), tensile strength (15.61 ± 0.43), folding endurance (160 ± 9), and drug release (84.98 ± 2.14) at 40 ± 0.5 °C/75 ± 5% RH till 6 months. At visual examination, the microbial growth was not observed on the surface of dressing. 4. Conclusion Wound healing is a major health care challenge. Dressing changes are uncomfortable for patients, which also causes a major financial and logistical burden. Therefore, an attempt was made to formulate moxifloxacin-loaded beads incorporated into spongy wound dressing with an objective to heal chronic wounds to reduce dressing change frequency. Dressing allowed slow release of drug through beads at wound area. Spongy dressing absorbed wound exudates and permeated water vapour transmission, required for fast heal. Thus, porous dressing delivered moxifloxacin on surface of wound with slow release, while preventing its penetration inside. Novel wound dressing achieved more contact time with wound area, easy applicability, less pain to the patient due to reduce frequency of dressing change. Ease of applicability at the wound area without any pain; reduced frequency of changing wound dressing with less pain to patients, ultimately, avoiding the chances of amputation, is major breakthrough at wound care system. Declaration of Competing Interest Authors declare that there are no conflicts of interest. Acknowledgement Authors are grateful to Macleods Pharmaceuticals, India for gifting moxifloxacin and Alfa Exim, India providing gift sample of CMTSP.
[1] J. Boateng, O. Catanzano, Advanced therapeutic dressings for effective wound healing—a review, J. Pharm. Sci. 104 (2015) 3653–3680, https://doi.org/10.1002/ jps.24610. [2] P.G. Bowler, B.I. Duerden, D.G. Armstrong, Wound microbiology and associated approaches to wound management, Clin. Microbio. Rev. 14 (2) (2001) 244–269, https://doi.org/10.1128/CMR.14.2.244–269.2001. [3] K. Vowden, P. Vowden, Wound dressings: principles and practice, Surgery (Oxford) 32 (2014) 462–467. [4] P.L. Bigliardi, S.A.L. Alsagoff, H.Y. El-Kafrawi, J.K. Pyon, C.T. Cheuk Wa, M.V. Anthony, Povidone iodine in wound healing: a review of current concepts and practices, Int. J. Surgery. 44 (2017) 260–268, https://doi.org/10.1016/j.ijsu.2017.06.073. [5] N. Mayet, Y. Choonara, P. Kumar, L. Tomar, C. Tyagi, D. Toit, V. Pillay, A comprehensive review of advanced biopolymeric wound healing systems, J. Pharm. Sci. (2014) 2211–2230, https://doi.org/10.1002/jps.24068. [6] T. Phaechamud, P. Issarayungyuen, W. Pichayakorn, Gentamicin sulfate-loaded porous natural rubber films for wound dressing, Int. J. Biol. Macromol. 85 (2016) 634–644, https://doi.org/10.1016/j.ijbiomac.2016.01.040. [7] S. Anjum, A. Arora, M.S. Alam, A.B. Gupta, Development of antimicrobial and scar preventive chitosan hydrogel wound dressings, Int. J. Pharm. 508 (2016) 92–101. [8] B. Singh, L. Varshney, S. Francis, Rajneesh, Designing tragacanth gum based sterile hydrogel by radiation method for use in drug delivery and wound dressing applications, Int. J. Biol. Macromol. 88 (2016) 586–602, https://doi.org/10.1016/j.ijbiomac. 2016.03.051. [9] R. Fu, C. Li, C. Yu, H. Xie, S. Shi, Z. Li, Q. Wang, L. Lu, A novel electrospun membrane based on moxifloxacin hydrochloride/poly(vinyl alcohol)/sodium alginate for antibacterial wound dressings in practical application, Drug Deliv 23 (2016) 828–839, https://doi.org/10.3109/10717544.2014.918676. [10] M. Tummalapalli, M. Berthet, B. Verrier, B.L. Deopura, M.S. Alam, B. Gupta, Composite wound dressings of pectin and gelatin with aloe vera and curcumin as bioactive agents, Int. J. Biol. Macromol. 82 (2016) 104–113, https://doi.org/10.1016/j.ijbiomac. 2015.10.087. [11] M.H. Asfour, H. Elmotasem, D.M. Mostafa, A.A. Salama, Chitosan based Pickering emulsion as a promising approach for topical application of rutin in a solubilized form intended for wound healing: in vitro and in vivo study, Int. J. Pharm. 534 (2017) 325–338, https://doi.org/10.1016/j.ijpharm.2017.10.044. [12] W.Y. Ng, A. Migotto, T.S. Ferreira, L.B. Lopes, Monoolein-alginate beads as a platform to promote adenosine cutaneous localization and wound healing, Int. J. Biol. Macromol. 102 (2017) 1104–1111, https://doi.org/10.1016/j. ijbiomac.2017.04.094. [13] E.E. Ozseker, A. Akkaya, Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads, Carbohydr. Polym. 20 (2016) 441–451, https://doi.org/10.1016/j.carbpol.2016.05.073. [14] A. Osmokrovic, I. Jancic, J. Vunduk, P. Petrovic, M. Milenkovic, B. Obradovic, Achieving high antimicrobial activity: composite alginate hydrogel beads releasing activated charcoal with an immobilized active agent, Carbohydr. Polym. 15 (2018) 279–288, https://doi.org/10.1016/j.carbpol.2018.05.045. [15] J. Qu, X. Zhao, Y. Liang, T. Zhang, P. Ma, B. Guo, Antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing, Biomaterials 183 (2018) 185–199, https://doi.org/ 10.1016/j.biomaterials.2018.08.044. [16] X. Zhao, H. Wu, B. Guo, R. Dong, Y. Qiu, P. Ma, Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing, Biomaterials 122 (2017) 34–47, https://doi.org/10.1016/j.biomaterials.2017.01.011. [17] J. Li, C. Jiang, X. Lang, M. Kong, X. Cheng, Y. Liu, C. Feng, X. Chen, Multilayer sodium alginate beads with porous core containing chitosan based nanoparticles for oral delivery of anticancer drug, Int. J. Biol. Macromol. 85 (2016) 1–8, https://doi.org/10. 1016/j.ijbiomac.2015.12.064. [18] T. Agarwal, S.N. Narayana, K. Pal, K. Pramanik, S. Giri, I. Banerjee, Calcium alginatecarboxymethyl cellulose beads for colon-targeted drug delivery, Int. J. Biol. Macromol. 75 (2015) 409–417, https://doi.org/10.1016/j.ijbiomac.2014.12.052. [19] D. Bansal, A. Gulbake, J. Tiwari, S.K. Jain, Development of liposomes entrapped in alginate beads for the treatment of colorectal cancer, Int. J. Biol. Macromol. 82 (2016) 687–695, https://doi.org/10.1016/j.ijbiomac.2015.09.052. [20] A. Pandit, G. Divase, T. Chavan, K.R. Khandelwal, Oral lipid based multiparticulate pastilles: design and effect of pore former, J. Pharm. Investig. (2015) 23–33, https://doi.org/10.1007/s40005-014-0141-4. [21] X. Zhao, P. Li, B. Guo, P. Ma, Antibacterial and conductive injectable hydrogels based on quaternized chitosan-graft-polyaniline/oxidized dextran for tissue engineering, Acta Biomater. 26 (2015) 236–248, https://doi.org/10.1016/j. actbio.2015.08.006. [22] N. Sanchez-Ballester, I. Soulairol, B. Bataille, T. Sharkawi, Flexible heteroionic calcium-magnesium alginate beads for controlled drug release, Carbohydr. Polym. 1 (2019) 224–229, https://doi.org/10.1016/j.carbpol.2018.11.096. [23] A. Pandit, S. Patel, V. Bhanushali, V. Kulkarni, V. Kakad, Nebivolol loaded microsponge gel for healing of diabetic wound, AAPS PharmSciTech 18 (2017) 846–854, https://doi.org/10.1208/s12249-016-0574-3. [24] N. Ninan, M. Muthiah, N. Kalarikkal In-KyuPark, A. Elain, T. Wong, S. Thomas, Y. Grohens, Pectin/carboxymethyl cellulose/microfibrillated cellulose composite scaffolds for tissue engineering, Carbohydr. Polym. 15 (2013) 877–885, https://doi. org/10.1016/j.carbpol.2013.06.067. [25] M. Buriuli, W. Kumari, D. Verma, Evaluation of hemostatic effect of polyelectrolyte complex-based dressings, J. Biomater. Appl. 32 (2017) 638–647, https://doi.org/ 10.1177/0885328217735956.
A.P. Pandit et al. / International Journal of Biological Macromolecules 140 (2019) 1106–1115 [26] N. Devi, J. Dutta, Preparation and characterization of chitosan-bentonite nanocomposite films for wound healing application, Int. J. Biol. Macromol. 104 (2017) 1897–1904, https://doi.org/10.1016/j.ijbiomac.2017.02.080. [27] L. Bennison, C. Miller, R. Summers, A. Minnis, G. Sussman, W. McGuiness, The pH of wounds during healing and infection: a descriptive literature review, Wound Practice and Research vol. 25 (2017) 63–69. [28] K. Kathe, H. Kathpalia, Film forming systems for topical and transdermal drug delivery, Asian J. Pharm. Sci. 2 (2017) 487–497, https://doi.org/10.1016/j.ajps.2017.07. 004. [29] J.S. Boateng, H.V. Pawar, J. Tetteh, Polyox and carrageenan based composite film dressing containing anti-microbial and anti-inflammatory drugs for effective wound healing, Int. J. Pharm. 441 (2013) 181–191, https://doi.org/10.1016/j. ijpharm.2012.11.045. [30] M.D. Romic, M.S. Klaric, J. Lovric, I. Pepic, B.C. Cizmek, J.F. Grcic, A. Hafner, Melatoninloaded chitosan/Pluronic® F127 microspheres as in situ forming hydrogel: an innovative antimicrobial wound dressing, Eur. J. Pharm. Biopharm. 107 (2016) 67–79, https://doi.org/10.1016/j.ejpb.2016.06.013. [31] A. Shrivastav, A.K. Mishra, S.S. Ali, A. Ahmad, M.F. Abuzinadah, N.A. Khan, In vivo models for assessment of wound healing potential: a systematic review, Wound Med 20 (2018) 43–53.
1115
[32] S. Murthy, M.K. Gautam, Shalini Goel, V. Purohit, H. Sharma, R.K. Goel, Evaluation of in vivo wound healing activity of Bacopa monniera on different wound model in rats, BioMed Res. Int 972028 (2013) 9, https://doi.org/10.1155/2013/972028. [33] S. Mahapatra, S. Mohanta, A. Nayak, Preliminary investigation of the angiogenic potential of Ziziphus oenoplia root ethanolic extract using the chorioallantoic membrane model, ScienceAsia 37 (2011) 72–74, https://doi.org/10.2306/ scienceasia1513-1874.2011.37.072. [34] N. Pettinelli, S. Rodríguez-Llamazares, V. Abella, L. Barral, R. Bouza, Y. Farrag, F. Lago, Entrapment of chitosan, pectin or κ-carrageenan within methacrylate based hydrogels: effect on swelling and mechanical properties, Mater. Sci. Eng. C Mater. Biol. Appl. 96 (2019) 583–590, https://doi.org/10.1016/j.msec.2018.11.071. [35] F. Shamekhi, E. Tamjid, K. Khajeh, Development of chitosan coated calcium-alginate nanocapsules for oral delivery of liraglutide to diabetic patients, Int. J. Biol. Macromol. 120 (2018) 460–467, https://doi.org/10.1016/j.ijbiomac.2018.08.078. [36] E. Salmerón-González, E. García-Vilariño, A. Ruiz-Cases, A. Sánchez-García, J. GarcíaSánchez, Absorption capacity of wound dressings: a comparative experimental study, Plast. Surg. Nurs. 38 (2018) 73–75, https://doi.org/10.1097/PSN. 0000000000000218.