Development of antibiotic and debriding enzyme-loaded PLGA microspheres entrapped in PVA-gelatin hydrogel for complete wound management

Abstract

A biocompatible moist system was developed for effective and complete wound healing. Optimized PLGA microspheres of gentamicin (GM) and serratiopeptidase (STP) were incorporated into PVA-gelatin slurry and casted into films to prepare multiphase hydrogel. The prepared system was characterized by in vitro and in vivo studies. Results revealed the uniform dispersion of microspheres in three-dimensional matrix of the hydrogel. The in vitro release data showed a typical biphasic release pattern. All parameters such as wound contraction, tensile strength, histopathological and biochemical parameters were observed significant (p < 0.05) in comparison to the control group. Results suggested an accelerated re-epithelialization with minimum disturbance of wound bed.

Keywords::

• Multiphase hydrogel
• wound healing
• serratiopeptidase
• gentamicin
• microspheres
• moist healing

Introduction

Wound healing is a highly intricate process involving sequential events beginning at the moment of injury and can continue for months to years (Hunt et al. 2000). The normal healing trajectory is delayed by the presence of infection and necrotic mass at the wound site, making the process more complex (Steel et al. 2006). Antibiotics offer effective therapy against the microbes in vitro, but intracellular localization of bacterium during infection makes the treatment difficult (Prior et al. 2004). This condition is further worsened in delayed wounds due to the extravasation of fibrous material into the wound site, limiting the therapeutic benefits of local or systemic antibiotics. In such cases, debridement becomes essential in the removal of dead and senescent cells that stand in the way of healing as tissue necrosis can be life-threatening. Among various methods, enzymatic hydrolysis by proteases is the most efficient, selective, and least traumatic means of dissolving this coagulum (Foster 2010). However, these bioactive agents exhibit increased biochemical and structural complexity, while repeated and high-dose administration of these agents causes toxicity, limiting their therapeutic potential. This necessitates an effective formulation design for the controlled co-delivery of enzymes and antibiotics in order to achieve effective tissue repair.

Drugs selected from each class were broad spectrum, antibiotic-gentamicin sulphate (GM) and protease- serratiopeptidase (STP). Gentamicin sulphate is a hydrophilic aminoglycoside antibiotic with a short half-life of 2–4 hrs used in the treatment of serious microbial infections (Rosenkrantz et al. 1980). Serratiopeptidase (STP) offers a powerful treatment for pain and inflammation with widespread use in wound debridement, arthritis, fibrocystic breast disease, chronic bronchitis, sinusitis, atherosclerosis, and carpal tunnel syndrome (Majima et al. 1990, Rawat et al. 2008). Moreover, serratiopeptidase has been reported to enhance antibiotic efficacy due to its antibiofilm property (Mecikoglu et al. 2006). In our previous studies, biodegradable PLGA [poly (D,L-lactic-co-glycolic acid)] microspheres of serratiopeptidase (Singh et al. 2009) and gentamicin (Singh et al. 2010) were prepared and optimized. Selected microspheres from previous studies were entrapped into the PVA-gelatin hydrogels to prepare a multiphase hydrogel system for better and faster wound healing.

Gel-forming polymers, e.g. poly (vinyl alcohol) (PVA), are of interest in wound healing as these drug delivery systems can provide soft, permeable, and hydrophilic interfaces with body tissues. Moreover, they exhibit features of moist wound healing with good fluid absorbance and are transparent to allow the monitoring of healing. The close resemblance of PVA hydrogels to human soft tissue (high water content, rubbery and elastic nature) has made them a material of choice for many biomedical applications, such as intervertebrate disc nuclei, artificial articular cartilage, contact lenses, matrices for cell immobilization, and mucoadhesives, as well as for the controlled release of drugs, growth factors, and proteins (Stauffer and Peppas 1992, Lee and Mooney 2001).

In this capacity, PVA hydrogels have been used as drug delivery vehicles, alone and in combination with other delivery systems, to provide desirable release profiles (DeFail et al. 2006). Gelatin is a multifunctional biopolymer and its presence in PVA solution enhances the processes of hydrogen bond formation, polymer crystallite formation, and phase separation, respectively, yielding a highly elastic gel (Mishra and Chaudhary 2010).

Drug release rates from PVA hydrogels tend to be relatively rapid (minutes to weeks) depending on the pore size, extent of cross-linking, and the nature of the incorporated drug and typically follow first-order kinetics (Bourke et al. 2003). To overcome this limitation, other controlled-release delivery systems (e.g., microspheres, nanoparticles, liposomes) can be entrapped within these PVA hydrogels without any detrimental effects that could occur in the presence of organic solvents and chemical cross-linking agents. The incorporation of microspheres and other particulate delivery systems can have advantages, such as isolation of the drug, slower drug release rates, and achievement of different drug release profiles, as well as incorporation of multiple drugs in different microsphere populations. Microspheres usually exhibit an initial rapid ‘‘burst’’ release as a result of the surface- associated drug (Kim and Burgess 2001). Furthermore, microsphere systems based on the commonly used poly(lactic- co-glycolicacid) (PLGA) polymer usually display a ‘‘triphasic’’ release profile, with an initial burst, followed by a minimal release phase, before entering into an approximate superlinear release profile when sufficient polymer degradation has occurred (Kim and Burgess 2001).

Although various systems have been prepared containing single drug-loaded microspheres or multiple drug-loaded scaffolds, based on the literature cited no work has been reported to date for co-delivery of STP and GM through PVA-gelatin hydrogels for complete wound healing. We aimed to develop a biocompatible system of PVA-gelatin hydrogel capable of carrying multiple drugs, STP and GM loaded in PLGA microspheres, for sequential delivery of multiple agents that act at multiple levels for effective and complete wound healing.

Materials and Methods

Materials

Serratiopeptidase (MW 52kDa) was received as a kind gift from Advanced Enzyme Technologies Ltd, Nasik, India. Gentamicin sulphate (GM) was received from RS Spectra Chemicals Ltd., Ahmednagar, Maharashtra, India, and PLGA with molar ratio of 50:50 (SGSITS, Indore). Poly vinyl alcohol (degree of hydrolysis 98.8%, mol. wt. 70000 Da) was purchased from Merck, India, and gelatin (for bacteriological purposes) was supplied by Loba Chemie, India, and used as received. All the other chemicals were of analytical reagent grade and triple distilled water was used in the preparation of all solutions.

Preparation of STP and GM loaded PLGA microspheres

STP and GM loaded PLGA microspheres were prepared by modified double-emulsion solvent evaporation technique following a protocol previously described (Singh et al. 2009, Singh et al. 2010). Briefly, STP and GM were dissolved in Tween® 20 (3% w/v) aqueous solutions. The internal aqueous phase (IAP) (0.5 ml) was emulsified with 5ml of organic phase containing PLGA (drug: polymer ratio of 1:3) by mechanical stirring for one minute (2000 rpm) (laboratory stirrer, 1NL-2116, Remi Motors Ltd., Mumbai, India). The temperature was maintained at 4°C using an ice bath. The organic phase consisted of dichloromethane: ethanol: isopropyl alcohol in a ratio of 5:6:4. The resulting primary emulsion (W₁/O) was added drop by drop to the volume of external aqueous phase (EAP) (100 ml) of 0.1% v/v Tween 80® solution. The aqueous Tween® 80 solution acts as an emulsion stabilizer. Emulsification was continued by stirring at 1200 rpm for five minutes to form multiple emulsion (W₁/O/W₂). The resulting (W₁/O/W₂) emulsion was stirred at room temperature (RT) for 5 8 h for solvent evaporation. The collected microspheres were washed three times with demineralized water (about 500 ml) by centrifugation at 10,000 g for 10 minutes. The microspheres were resuspended in distilled water and lyophilized for 24 hrs (Heto power dry LL 3000 Lyophilizer). The final product was stored in a dessicator at 2 8°C.

Morphological characterization

The microspheres were examined morphologically by optical microscopy (Erma, Tokyo, Japan) and scanning electron microscopy (SEM). Microspheres and hydrogels were mounted on the standard specimen mounting stubs and were coated with a thin layer of gold by a sputter-coating unit (VG Microtech, Uckfield, UK). Microspheres and hydrogels were viewed using a Leo (VP-435, Cambridge, UK) scanning electron microscope operated at an accelerating voltage of 15 kV.

Preparation of PVA-gelatin hydrogel

PVA-gelatin hydrogel was prepared by the freeze-thaw method with slight modifications reported by Michael et al. (2007) and Cascone et al. (2004). In a typical experiment, 10% PVA aqueous solution was prepared by adding 10g of PVA in 100ml of hot water with stirring at 80°C for 20 min. Aqueous solutions of gelatin (Gel) were prepared, adding 2.5g of gelatin to 100ml of distilled water under stirring at about 50°C. The PVA solution was blended with the gelatin to produce Gel/PVA blends with 20/80 as weight ratios H2 (optimized through preliminary studies with Gel/PVA blends H1, H2 and H3 with 10/90, 20/80, 30/70 weight ratios, respectively, shown in Table I).

Table I. Preliminary studies for selection of gelatin-PVA blend H1, H2, and H3 with 10/90, 20/80, 30/70 weight ratios, respectively.

S. No	Studies	Methodology	Results
1.	Infra-red spectroscopy	FTIR of PVA, gelatin and PVA-gelatin Hydrogel were taken (Schimadzu FTIR-8400S, Tokyo, Japan)	FTIR of hydrogel showed the peak due to C = O stretching of the gelatin at 1680 cm^–1 shifted to 1760 cm^–1 in the PVA-gelatin blend. No peaks were observed at 1680 cm^–1 indicating esterification of all the free carboxylic groups of gelatin in the blend.
2.	Scanning Electron Microscopy	The surface morphology was investigated by scanning electron microscopy (Leo, VP-435, Cambridge, UK).	Pure PVA hydrogel showed porous morphology. PVA-gelatin blend showed lamellar appearance and an apparent preferential orientation in ratio of 20/80 (H2), but on further increase it became less ordered.
3.	Water vapor transmission rate (WVTR)	WVTR was measured using the upright wet cup method as described by ASTM E96.	The PVA-gelatin films showed “wet” WVTR characteristics that are close to that of intact skin. H1 and H2 had almost similar transpiration rates at 1308.59 g/m^2/24 h and 1386.71 g/m^2/24 h, respectively. H3 transmitted water vapor significantly faster at 1445.31 g/m^2/24 h.
4.	Hemocompatibility	The hemocompatibility tests were carried out broadly on the basis of ASTM standard.	H2 was (20/80) found to be highly hemocompatible with % hemolysis less than 5% as compared to other blend ratios.
5.	Water uptake capacity	A conventional gravimetric procedure was followed	Swelling ratio for PVA-gelatin blends H1, H2, and H3 were found to be 1.24 ± 0.23, 1.28 ± 0.33, and 1.32 ± 0.38, respectively.
6.	Diffusion coefficient	A diaphragm cell was used to measure the diffusion coefficient	The diffusion coefficient of drugs (GM and STP) through the membrane was found to be 1.42 × 10 ^− 5 cm^2/s and 1.35 × 10 ^− 5 cm^2/s, respectively, in the case of H2.

Incorporation of microspheres in hydrogels

Pure drug-containing hydrogels (PDH) were prepared by adding a calculated amount of drugs (equivalent to STP 100mg and GM 100mg) into the hydrogel. Similarly, an accurately weighed amount of drug-loaded PLGA microspheres were dispersed in a PVA-gelatin slurry, followed by homogenization at 1000 rpm for 20 min for uniform distribution (DPMH). A total of 10ml of respective solution was placed in a petri dish (internal diameter 10cm, Corning) and kept for freeze-thaw cycles. The samples underwent five freeze-thawing cycles to obtain hydrogels. Each cycle, with the exception of the first one, consisted of 1h at − 20°C and 30 min at room temperature. The first cycle differed from the others due to a longer standing time (overnight) at − 20°C. The prepared hydrogels were washed with triple distilled water and the petridish was sealed with the parafilm and kept under U.V. light for sterilization. The prepared hydrogel films were used for subsequent characterization and in vitro release studies.

In vitro release studies

The drug-loaded PLGA microspheres (MS) and MS loaded PVA-gelatin hydrogels were placed in contact with 10ml of PBS (pH 7.4). Suspension was taken in a hollow glass tube of one inch diameter, which was tied at one end with a treated cellophane membrane. This tube was clamped in 100 ml of PBS (pH 7.4) and maintained at 37 ± 2 °C on a hot plate magnetic stirrer. At each time point, the samples withdrawn from the recipient compartment were analyzed for drug content by simultaneous methods of estimation using first derivative spectra at 229 nm and 339 nm (derivatized with o-phthaldialdehyde) for STP and GM, respectively. The samples were refreshed with fresh PBS (Singh et al. 2009, Singh et al. 2010). All the in vitro release studies were conducted in triplicate (independently prepared films and different batches of microspheres), and mean values and standard deviations were calculated.

In vivo studies

A full thickness wound model was selected to evaluate the performance of the prepared delivery system. All studies were conducted according to the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), Government of India, and permission was taken from the institutional animal ethical committee (registration no 600/01/a/CPCSEA/2001). The experiments were carried out on random-bred Swiss albino mice maintained in the animal house of the Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal, M.P. The animals were acclimatized to laboratory hygienic conditions for 10 days before starting the experiment with controlled conditions of light (12:12, light and dark), temperature (22 ± 3°C), and humidity (50 ± 10%). An excision wound was inflicted by cutting away a 500mm² full thickness of skin from a predetermined area; the wound was left undressed to the open environment.

Grouping of animals

The selected animals (mice) were divided into three groups of six animals in each group. Group I (control group) consisted of animals treated topically with prepared 20/80 blend PVA hydrogel (H2) (without any drug). Group II consisted of animals treated topically with PDH formulation (hydrogel containing pure drug STP and GM). Group III consisted of animals treated topically with DPMH formulation (multi-phase hydrogel-containing PLGA microspheres of STP and GM).

Wound contraction and epithelialization time

The percentage wound contraction was determined by measuring the area of the wound by a tracing method using transparent paper and the area measured by graph paper. Wounds were measured regularly as per the decided schedule in an interval of five days until complete healing, using the following formula:

The epithelialization time was measured from the initial day.

Hydroxyproline (collagen estimation) and protein (bovine serum albumin) measurement

Tissues were dried in a hot air oven at 60–70°C to constant weight and were hydrolyzed in 6N HCl at 130°C for 4 h in sealed tubes. The hydrolysate was neutralized to pH 7.0 and was subjected to Chloramine-T oxidation for 20 min. The reaction was terminated by addition of 0.4M perchloric acid and color was developed with the help of an Ehrlich reagent at 60°C and measured at 557 nm using a spectrophotometer (Woessner 1961). From the collected tissue, protein estimation was done using copper and the Folin reagent.

Histopathological study

Animals were anesthetized before taking a skin sample on corresponding postoperative days.

Wound tissue specimens were taken after every five-day interval until complete healing from Group I (4, 8, 12, and 16 days), Group II (4, 8, 12, and 16 days), and Group III (4, 8, and 12 days as complete healing was observed on day 12). Wound tissue specimens were collected from all groups and, after the usual processing, 6-mm-thick sections were cut and stained with haematoxylin and eosin (McManus and Mowry 1965). Sections were qualitatively assessed under the light microscope and observed for fibroblast proliferation, collagen formation, angiogenesis, and epithelialization.

Statistical analysis

Groups I, II, and III were compared. The results were analyzed statistically using Student's t-test to identify the differences between the treated and control. The data were considered significant at p < 0.05.

Results and Discussion

Preparation and characterization of PLGA microspheres

The STP and GM loaded PLGA microspheres were prepared using a double emulsion technique and then examined using SEM. The average microsphere sizes of STP and GM loaded systems were 25.54µm and 22.56µm with entrapment efficiency of 75.86% and 85.35%, respectively. The microspheres were spherical and exhibited a smooth surface morphology (Figure 1a, b). Microspheres were evaluated for stability by studying morphological changes as we intended to prepare a system meant for sustaining the drug release for wound management with minimum disturbance of the wound bed. Microspheres exhibited significant morphological changes. The surface of the microspheres appears to be rougher and wrinkled by day 12 without loss of spherical morphology (Figure 1c, d). However, after 21 days, the shape became less spherical with more evident micropores and loss of the integrity of the spherical structure (Figures 1c, d, e, and f).

Figure 1. Scanning electron micrographs of microspheres; a) STP-loaded PLGA microspheres; b) GM-loaded PLGA microspheres; c) drug-loaded PLGA microspheres on day 12 at high magnification (STP); d) drug-loaded PLGA microspheres on day 12 at high magnification (GM); e) drug-loaded MS after 21 days at high magnification (STP); f) drug-loaded MS after 21 days at high magnification (GM).

In our previous studies, microspheres were optimized in terms of polymer content (PLGA) and external aqueous phase volume (EAP) in order to get maximum entrapment so that the drug could be sustained for a long period of time (Figure 2). Response surface graphs indicate the positive effect of PLGA concentration on mean diameter of microspheres with little insignificant effect of EAP. This was attributed to increase in polymer viscosity with increase in polymer concentration leading to increased size of microspheres. EAP significantly affected entrapment efficiency (Singh et al. 2009, Singh et al. 2010).

Figure 2. 3D surface curve for the effect of selected variables for desirability in terms of maximum entrapment and particle size in range.

Preparation and characterization of hydrogel

The PVA solution was blended with the gelatin to produce Gel/PVA blends with the following weight ratios: 10/90, 20/80, and 30/70 coded as H1, H2, and H3, respectively. The prepared hydrogels were characterized by FTIR, Scanning Electron Microscopy (SEM), water vapor transmission rate (WVTR), hemocompatibility, water uptake capacity, and diffusion coefficient. All the data relating to preliminary investigations on PVA-gelatin hydrogel are given in Table I. We selected H2 (20/80) for further studies as its physiological parameters were comparable and closer to skin for suitability as wound-dressing material.

The hydrogels were prepared by physical cross-linking due to crystallite formation without requiring any cross- linking agent. Such physically cross-linked materials also exhibit higher mechanical strength than PVA gels cross-linked by chemical or irradiative techniques because the mechanical load can be distributed along the crystallites of the three-dimensional structure (Hassan and Peppas 2000).

Gelatin is a multifunctional biopolymer and it was incorporated with PVA solution to yield a highly elastic gel by the processes of enhanced hydrogen bond formation, polymer crystallite formation, and phase separation. Hydrogels produced by the freezing-thawing method were porous in nature, which may be due to the fact that the freezing of a PVA-gelatin mixture results in formation of ice crystal domains within the polymer mixture matrix and the thawing process results in melting of the ice crystal, thus leaving wide pores in the gel.

IR spectral analyses were carried out to confirm the formation of the PVA-gelatin network (Table I). IR spectra indicated the formation of esterified product with an ester linkage, a secondary alcoholic group, and secondary amide groups in addition to the hydrocarbon chromophore (spectra not shown).

The prepared PVA-gelatin films showed wet WVTR characteristics that are close to those of intact skin, potentially capable of both transpiring a substantial amount of excess exudates and maintaining occlusivity to retain the therapeutic quantity of wound moisture. The addition of gelatin can greatly improve the hydrophilicity of PVA. Moreover, both gelatin and PVA have film-forming ability; thus the hydrogel blend could be a good candidate as artificial skin for complete wound management. An intermediate WVTR was needed, as an ideal wound dressing must control the water loss from a wound at an optimal rate. Hemocompatibility, water uptake capacity, and diffusion coefficient were further investigated and supported the selection of 20/80 Gel/PVA. Thus 20/80 Gel/PVA hydrogels were selected for further experimentation.

Incorporation of microspheres in hydrogels

The photomicrograph of the microsphere-embedded hydrogel is presented in Figure 3. Opaque microspheres were visible in the transparent hydrogel network, forming a new system of microsphere in the hydrogel (Figure 3a, b). SEM revealed the uniform dispersion of microspheres in a three-dimensional lamellar matrix of the PVA-gelatin hydrogel formulation (Figure 3c).

Figure 3. Multiphase hydrogel: (a) hydrogel film; (b) photomicrograph of hydrogel film; (c) SEM of microsphere-loaded PVA-gelatin hydrogel.

Common hydrogels are not good for uniform incorporation of particles in their 3D matrix. Therefore, the freeze-thaw method was employed for the incorporation of particles into a gel matrix. All the formulations had a random morphology and similar structure.

In vitro release studies

The in vitro release of drugs (STP and GM) from the PLGA microspheres and the drug-loaded PLGA microspheres in PVA-gelatin hydrogels (DPMH) were studied. The in vitro release data showed that the release of drugs entrapped in microspheres from hydrogels had a typical biphasic release pattern, i.e., a burst release followed by a slower sustained release (Figure 4).

Figure 4. Cumulative release of gentamicin (GM) from PLGA microspheres (▪) and serratiopeptidase (STP) from PLGA microspheres (+); GM incorporated into the PVA-gelatin hydrogel matrix GPH (♦); STP incorporated into the PVA-gelatin hydrogel matrix SPH (-); pure STP and GM loaded into hydrogels (▴) and (×), respectively (results are given as M ± SD (p < 0.05), n = 3, where n is the number of repeat experiments conducted).

Within one day, the microspheres demonstrated a high burst release (70.2 ± 3.56 and 69.32 ± 5.31% for GM and STP, respectively). The burst release was delayed in hydrogels incorporating the microspheres. Drug-loaded microspheres loaded onto the hydrogel exhibited a similar burst release on day 8. The majority of the STP and GM loaded microspheres on hydrogel showed around 80% release by day 12. The percent cumulative release of STP and GM from the microspheres was statistically higher than the release from the PVA-gelatin hydrogels containing STP and GM loaded microspheres at every time point (p < 0.01). This evidence demonstrates that incorporating the microspheres into PVA-gelatin hydrogel delays the burst effect commonly seen in the release of bioactives from microspheres alone (Galeska et al. 2005, Bajpai and Bhanu 2004). Drugs entrapped in microspheres completely released the drug in four days whereas microsphere (MS) loaded hydrogels sustained the release for 20 days, signifying the extended drug release without disturbance of wound bed to be suitable as wound dressing. Plain drugs loaded into hydrogels released completely in two days.

The possible mechanism of delayed drug release from PLGA microspheres involves dramatic decrease in the acid-catalyzed, self-accelerated ester bond cleavage. PLGA degradation leads to a build-up of acidic oligomeric by-products of PLGA, which prefer to remain within the non-polar environment of the PLGA microspheres rather than into aqueous release media (Li et al. 1990a, Li et al. 1990b). Possible decreased release of drugs from the MS loaded hydrogels could be due to the formation of microcrystalline PVA domains at the PLGA microsphere surfaces, significantly decreasing the ability of water to partition into the microspheres with decrease in polymer degradation and drug release (DeFail et al. 2006).

The release rates were analyzed by the least square linear regression method. Release models such as the first order model, Higuchi model, and Ritger-Peppas empirical model were applied to the release data.

The coefficient of determination (R²) of equation for release of drugs from microsphere incorporated hydrogels (GPH and SPH) from all microspheres in phosphate buffer was > 0.9, signifying a first order release pattern. The value of coefficient of determination (R²) in the Higuchi equation was also found to be > 0.9, which indicates the integrity of the PVA-gelatin hydrogel and diffusion-controlled release. Substituting the release values in the Ritger-Peppas equation, the value of coefficient of determination was again about > 0.9.

The value of n obtained was found to be in the range of 0.431–0.775, except plain drug-containing hydrogels (GH and SH) (Table II). Hydrogel containing GM and STP microspheres (GPH, SPH) showed non-Fickian release as n > 0.43 but < 0.85 for non-Fickian (anomalous) release; n = 0.43 indicates Fickian (case I) release; and > 0.89 indicates super case II type of release. Non-Fickian refers to a combination of both diffusion and erosion controlled-drug release (Siepmann and Peppas 2001). This result was attributable to the sustained release of the drug, signifying a mixed type of release pattern.

Table II. Release behavior of drugs in phosphate buffer (pH 7.4) from hydrogel formulations.

Formulation code	First order		Higuchi		Ritger-Peppas
	k	R^2	k	R^2	n	R^2
GPH	2.252	0.978	17.74	0.984	0.446	0.982
SPH	2.203	0.957	17.99	0.967	0.431	0.960
GH	0.057	0.881	10.62	0.991	0.282	0.979
SH	0.057	0.907	10.47	0.996	0.271	0.981

*K, Release rate constant; R², coefficient of determination; n, release exponent.

In vivo studies

Healing is a physiological process and wounds cause discomfort and are prone to infection and other complications. Therefore, the use of agents expediting healing is indicated.

Epithelialization

The macroscopic analysis of the wound revealed that the DPMH-treated group took only 12 days for complete epithelialization; those groups treated with PDH required a total period of 16 days, whereas the control groups took 18 days for complete epithelialization (Table III). Complete recovery in the case of the untreated group (the wound was kept uncovered) showed complete healing in more than 30 days. It was observed that the healed area of the formulation-treated group was regular and uniform compared to that of the control groups (Group I).

Table III. Effect of formulation and standard preparation on % wound contraction and epithelialization period on excision wound model in mice.

Groups	Post-Wounding Days
	4	8	12	13	14	15	16	17	18	E.P
I-H2 (control) (without any drug)	53.34 ± 0.62	75.03 ± 0.54	83.34 ± 0.69	89.41 ± 0.85	94.12 ± 0.62	95.81 ± 0.84	96.08 ± 0.83	98.87 ± 0.65	99.67 ± 0.47	18
II-PDH hydrogel containing pure drug STP and GM)	55.39 ± 0.32	86.96 ± 0.45	93.68 ± 0.43	96.32 ± 0.65	97.9 ± 0.61	99.4 ± 0.73	100 ± 0.64	–	–	16
III-DPMH Multi-phase hydrogel containing PLGA microspheres of STP and GM)	65.89 ± 0.42	89.01 ± 0.49	100 ± 0.92	–	–	–	–	–	–	12

n = 6 albino mice per group;, tabular value represents Mean ± S.D; E.P-Epithelialization Period.

Wound contraction

An excision wound contraction was measured planimetrically using a transparent paper in each four-day interval up to 12days and afterwards by each day until complete healing was achieved. Results were noted and finally presented after statistical treatment (Table III).

The percent wound contraction was measured using the following formula:

Periodical observation of animals showed a significant increase in the rate of contraction in experimental groups when compared to the control. On day 12, the wounds dressed with DPMH (Group III) (multi-phase hydrogel containing PLGA microspheres of STP and GM) contracted completely against the control group (p < 0.01), which showed only 83.34 ± 0.69% of contraction (Figure 5a-i). The PDH-treated group (Group II) showed complete contraction on the sixteenth day, earlier than the control (complete healing took 18 days) with smooth skin and negligible scar formation, whereas on day 12 it showed 93.68 ± 0.43% contraction.

Figure 5. Photographs of wounds 4, 8, and 12 days after surgery illustrating the changes in wound contraction and epithelialization with TS of mice skin on the twelfth day in all groups stained with hematoxylin and eosin at 40 x (A) control; (B) serratiopeptidase and gentamicin in hydrogel; (C) serratiopeptidase and gentamicin PLGA microspheres in hydrogel.

The observed increase in the contraction of wounds in the animals treated with DPMH (experimental) is attributed to the increased rate of healing. Among the experimental groups, those treated with DPMH showed quick healing when compared to other groups. This may be attributed to the moist environment provided by the hydrogels promoting the formation of granular tissue while the combination of the antimicrobial and debriding agents assisted in producing a symbiotic physiological environment duly synergizing the healing process. Serratiopeptidase promotes the removal of a dry layer of dead and necrotic tissue. It generally helps with the healing process of injured tissues and reduces pain–inducing amines from the inflamed tissues. Moreover, enzymatic debridement of the collagen may act as a chemotactic factor, resulting in the attraction of fibroblast cells to the wound site, and in turn producing biomaterials for dermal repair (Rath et al. 2011).

Various studies have been carried out to establish the clinical role of serratiopeptidase as an effective analgesic, anti-inflammatory, and debriding agent. Khateeb and Nusair reported the significant role of proteolytic enzyme serrapeptase on reducing the swelling, pain, and trismus after surgical extraction of mandibular third molars (Al-Khateeb and Nusaira 2008). Recently, Rath et al. (2011) reported similar results with a combination of metronidazole- and serratiopeptidase-loaded alginate microspheres prepared by the emulsification method. Experimentation suggested improved wound healing using serratiopeptidase and metronidazole in full thickness wounds in rabbits. However, the effective period was less, requiring frequent disturbance of the wound bed.

Better and rapid healing was observed in group III (DPMH), which might be due to retention of activity of bioactive drugs entrapped within microspheres, further supported by our previous studies (Singh et al. 2009, Singh et al. 2010). Thus with combination therapy, contraction proceeds at a rapid rate with complete epithelial closure.

Hydroxyproline (collagen estimation) and protein (bovine serum albumin) measurement

Hydroxyproline content in the granulation tissues of control and experimental mice on different days after wound creation was significantly higher in the formulation-treated group. On the eighth day, the collagen level was found to elevate to about 1.5–1.6-fold in group III when compared to the control, while group II showed almost similar results to those of the control group. The significant increase in the collagen content of granulation tissue isolated from experimental groups may be due to an increased synthesis of collagen and could be correlated with the effective healing of wounds. A decreasing trend in the collagen content after day 10 in all the groups was observed. A lack of correlation between the contraction process and the total amount of collagen in the wound was evident. A linear increase in total protein content was observed in all groups, including the control. Group III again showed significant increased percentage of total protein.

Histological analysis

The processes involved in wound healing are epithelialization, contraction, and connective tissue deposition. The involvement of each phase varies over a spectrum dependent largely on the type, location, and milieu factors influencing the wound.

Histological studies are crucial to evaluate the healing of wounds. Keeping this in view, the histological sections taken from the biopsies were studied for the healing pattern of wounds in the control and experimental groups. All wounds showed more or less similar patterns (Figure 5).

The histological section of wound treated with DPMH (Group III) after 12 days (Figure 5CIII) revealed complete healing with the beginning of remodeling of the skin. From photographs it can be observed that the keratinocytes are clearly differentiated from the epidermal layer and are accumulated in the basal lamina of thick epidermis with rete ridges and a thick stratum corneum. Further, the collagen fiber occupied most of the wound granulation tissue.

In the case of control wounds, complete healing was observed on the eighteenth day. There were no significant differences between the histological scores of the wounds in groups I and II as less thick and flat epithelium without rete ridges was observed for the healed wound bed. Wound healing was accepted as complete when the wound area was totally covered by epithelization and contraction. All the wounds were healed in 20 days except the uncovered wound, where complete healing took more than 30 days. Early and better healing, even in the case of the control group as compared to the uncovered wound, could be due to the fact that the wounds were covered with the moist dressings, supporting the early granulation. The formulated hydrogels, apart from acting as a wound-covering material, also acted as occlusive dressings, thus providing a moist environment, which accelerates the migration of keratinocytes, induces exudation, and stimulates the development of granulation tissue. Wound-healing time in combination therapy involving microspheres was significantly reduced in comparison with other groups.

Conclusion

The developed delivery system is effective in promoting natural debridement by hydrating necrotic tissue with loosening and absorbing slough and exudate in wounds. It also encourages autolytic debridement, particularly useful for deeper wounds as it can be applied directly into the wound, maintaining a moist wound environment, allowing healing to occur from the base of the wound upwards, and encouraging rapid granulation and re-epithelialization. The present results suggest an accelerated re-epithelialization under the prepared hydrogel with good cosmetic effect. It provides a direct sustained release of the antimicrobial and debriding agent at the wound surface to provide a long-lasting antimicrobial action in combination with maintenance of a physiologically moist environment for healing with minimum disturbance of the wound bed.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.