An Appraisal of pH triggered Bacitracin drug release,through composite hydrogel systems

Hydrogel fabrication using solvent casting method

Comparison of the physical characteristics

In-Vitro Studies

Drug incorporation

Bacitracin zinc drug was loaded in hydrogels by permeation method. Hydrogel samples (1 × 1cm) of chosen gel ratios were placed in a filtered drug saturated solution, at a drug concentration of 34 mg Bacitracin Zinc in 10 mL PBS solution, (absorbance at 470 nm = 0.0250) for 60 min, keeping in view the maximum time taken by samples to reach the EWC. After 60 min, the samples were air dried. Gel drying and consequent changes in weight were monitored carefully. The concentration of drug per sample, as well as the physical characteristics of the hydrogels are given in (Table 1).

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Table 1.

Physical characteristics of all hydrogel composites along with concentration of drug (mg) per sample weight (mg).

CHITOSAN GELATIN ; HEMA GELATIN COMPOSITE HYDROGEL FILM FORMULATIONS (PHYSICAL PARAMETERS)
Film Ingredients			Physical Parameters
Chitosan (ml)	Gelatin (ml)	Hydrogel film	Thickness (mm)	Total Mass (g)	%EWC
6.67	13.33	1:2 (1x1 cm)	0.16 ± 0.01	0.019 ± 0.015	95
5	15	1:3 (1x1 cm)	0.17 ± 0.01	0.018 ± 0.013	94
4	16	1:4 (1x1 cm)	0.18 ± 0.01	0.018 ± 0.015	94

Film ingredients			Physical Parameters
HEMA	Gelatin	Hydrogel film	Thickness (mm)	Total Mass (g)	%EWC
5	15	1:3 (1x1 cm)	0.62 ± 0.01	0.48 ± 0.015	63
4	16	1:4 (1x1 cm)	0.59 ± 0.01	0.47 ± 0.013	69
3.33	16.67	1:5 (1x1 cm)	0.60 ± 0.01	0.49 ± 0.013	65

CONCENTRATION OF DRUG (mg) PER SAMPLE WEIGHT (mg)
Hydrogel Category	Total Mass (mg)	Bacitracin Zinc (mg)
CG Hydrogel (1x1 cm)	20	0.5 ± 0.011
HG Hydrogel (1x1 cm)	60	0.51 ± 0.010

It shows that 0.025 mg drug was incorporated in each mg of C/G sample and 0.0085 mg drug was absorbed by each mg of H/G sample. The incorporation of drug was further investigated through UV spectroscopy and IR spectroscopic techniques.

Material characterization

Cutaneous application tests for the hydrogel systems

For establishing an understanding of the preliminary potential cutaneous/topical applicability of the hydrogel dressings, a murine model was investigated, as these have been proven to be suitable with reference to initial in-vivo investigations. IRB approval was acquired before the in-vivo investigation that involved 12 Male BALB/c mice, at 8 weeks and 22–26 g (an average of 0.024 kg) in weight. Four experimental groups were used for the cutaneous applicability investigations.

Wound formation and treatment

Hair from the dorsal side of mice were removed using a depilatory cream. Local anesthesia of Ketamine and Xylazine cocktail (1:7) was administered in intraperitoneal cavity, according to the weight of mice (0.1 mL/20g). ⁴⁸ After confirming the unconsciousness of mice, their bare skin were wiped using alcohol swab and abrasion wound of 1 cm × 1 cm was created on each mouse, using sterile surgical blade. The desired wound area was marked prior to wound creation, and a calibrated scale was used to ensure area/measurement uniformity for all experimental and control groups. Group randomization was not carried out for this series of experiments.

The wounds of negative control group were left untreated and uncovered in accordance with the procedure outlined by El-ezz et al. ⁴⁹ The wounds in positive control group were covered with a commercially available ointment Polyfax (active ingredients Polymixin B Sulphate and Bacitracin Zinc). A thin layer over the wound area was applied and spread after addition of a 5 mm ribbon of ointment, which is equivalent to 100 mg of ointment, containing 0.68 mg of Bacitracin Zinc. The wounds of both experimental groups were shielded with their respective wound dressings, first drug loaded hydrogel; the periphery of the hydrogel dressings were held in place with surgical tape. For cutaneous application tests, a 1 cm × 1 cm film for both C/G and H/G groups was used, with a total weight of 20 mg and 60 mg respectively and a drug loading of 0.5–0.51 mg each (as depicted in Table 1). The pH and diameter of the wounds were measured with a calibrated scale and wound area was assessed daily for 5 days. Images were recorded at fixed magnification (Figure 9(a)–(C), Table 2) to monitor the wound condition. The wound dressings in both experimental groups were replaced every day and fresh ointment on the positive control group was applied, after wiping the wound with alcohol swabs. The negative control group received the same alcohol swab treatment on daily basis as compared to the other experimental groups. The results of all the experimental groups were compiled and compared with a semi-quantitative assessment of wound diameter, at the end of the experiment. Wound diameter percentage changes were mapped out, in accordance with previous published investigations ⁵⁰ of area based wound assessment through the equation

P e r c e n t a g e o f W o u n d D i a m e t e r R e d u c t i o n = ( [ W D i − W D f ] ÷ W D i ) ∗ 100

Where WDi denotes the initial wound area and WDf denotes the final wound diameter.

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Table 2.

Diameter reduction data for all experimental and control groups in the cutaneous applicability test model.

GROUPS	WOUND DIAMETER (mm)
		DAY 1	DAY 2	DAY 3	DAY 4	DAY 5
NEGATIVE CONTROL	1	10	10	10	9	9
	2	10	10	9	9	8
	3	10	9.5	9	8.5	8
	AVERAGE	10	9.83	9.33	8.83	8.33
	MEDIAN	10	10	9	9	8
	STD.P	0	0.2357	0.4714	0.2357	0.4714
POSITIVE CONTROL	1	10	9	9	8	6
	2	10	9	8	8	7
	3	10	9	8	7	6.5
	AVERAGE	10	9	8.33	7.67	6.5
	MEDIAN	10	9	8	8	6.5
	STD.P	0	0	0.4714	0.4714	0.40825
Chitosan/Gelatin EXPERIMENTAL GROUP	1	10	9	8	5	3
	2	10	9	8	6	4
	3	10	8.5	7	5.5	3
	AVERAGE	10	8.83	7.67	5.5	3.33
	MEDIAN	10	9	8	5.5	3
	STD.P	0	0.2357	0.4714	0.40825	0.4714
HEMA/Gelatin EXPERIMENTAL GROUP	1	10	9	7	5	3
	2	10	9	8	6.5	5
	3	10	9	7.5	6	4
	AVERAGE	10	9	7.5	5.83	4
	MEDIAN	10	8	7.5	5.83	4
	STD.P	0	0	0.408	0.62361	0.8165

Comparison of dimensional characteristics

Table 1 depicts the various measurements of C/G and H/G hydrogels respectively, in different ratios. The average values show that H/G films are a little thicker and heavier than C/G films, may be due to the greater number of ingredients used in their fabrication. Other than this observation, both films have uniform thickness and weight throughout and can make a suitable wound dressing conforming to the skin surface and is breathable as they are thinner than human skin.

Equilibrium water capacity study results

For 1:2, 1:3 and 1:4 of C/G compositions, the EWC percentage is 95%, 94% and 94% respectively. For 1:3, 1:4 and 1:5 of H/G compositions, the EWC percentage is 63%, 69% and 65% respectively.

Figures 2(a) and (b) represent Equilibrium Water Capacity results of both C/G and H/G hydrogels in three ratios each. On comparison of the results, it can be observed that C/G films have more swelling rate and water capacity than H/G films, even though they are thinner than H/G films. They allow more water uptake than H/G films and can absorb more exudate from the wound site.

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Figure 2.

Comparison of weight change in three C/G and H/G compositions in distilled water.

From the derived results, one ratio from each hydrogel was picked out for further studies, 1:3 of C/G and 1:4 of H/G, based on their maximum water capacity and highest time of degradation. EWC tests were carried out to chart out the aqueous absorbing capacity of the two hydrogel systems. The results of the EWC tests reveal, that the time taken for the two categories of hydrogels to reach full absorption capacity, is not similar. All C/G samples (regardless of the ratio tested) indicate an aqueous uptake of in approximately 60 min. Gravimetric changes for these hydrogels were mapped out over a time span of 80 min. It was observed that the C/G compositions 1:2 and 1:4 lost dry weight (indicating degradation) after a time period of 60 min 1:3 remained stable, and the weight of this sample remained constant, after 60 min. This observation could be attributed to the difference in crosslinking degree between the polymeric chains of the two components, Chitosan and Gelatin. With reference to C/G 1:3, the degree of cross-linking may potentially be optimal, thus the sample has greater mechanical integrity, even after it is subject various swelling pressures and osmotic changes. Considering this, C/G 1:3 was used for further experimental studies.

With reference to H/G ratios tested, it was observed that the hydrogel degradation point (shown as a drop in the gravimetric curve after a specific time span) came after a delayed time period, in case of H/G 1:4. EWC for this ratio was also the highest (69%) as compared to H/G 1:3 (63%) and H/G 1:5 (65%). The occurrence of amine groups (-NH₃) in both C/G and H/G gels mean that these systems are intrinsically, highly hydrophilic. The C-O-C linkage confers mechanical integrity to the individual hydrogel components. Even after swelling has occurred, the physical integrity of the hydrogels may be attributed to this linkage, facilitated by the Glutaraldehyde component.

It is also obvious from the equilibrium water capacity tests that for the same immersed volume of hydrogel samples, the EWC of HEMA/Gelatin samples is much lower as compared to Chitosan/Gelatin composite hydrogels. This could be attributed to the presence of the hydrophobic methacrylate groups in the HEMA backbone.

The EWC tests reveal information about the maximum potential of drug loading that these hydrogel systems can carry. Permeation based drug loading methods have been used to synthesize drug loaded hydrogel systems hence the quantity of dissolved solute absorbed, would ultimately reflect the final drug load capacity through immersion into a saturated drug solution. Hence it was feasible to carry out further experimentation on the hydrogel composite that indicates the highest EWC along with the most delayed degradation time. This high drug loading capacity would confer the hydrogel systems with a high drug dose (according to the requirement), a long degradation time, that could translate into extended usability in the clinical scenario. The C/G 1:3 composite and the H/G 1:4 composite were therefore tested further for drug loading and release testing. These hydrogel systems were also characterized further through spectroscopic analysis.

Comparison of drug release kinetics

The graphical interpretation of the results (Figure 3(a) and 3(b)) demonstrate the average amount of drug released at pH 4, 6, 7.4, 8 and 10 from both hydrogels over the timespan of 48 h. All three mechanisms of drug release were observed over time, ⁵¹ swelling at start when solution enters the hydrogel structure, then the embedded drug starts diffusing out of hydrogel into the solution. In both hydrogels, the drug release at lower (acidic) pH values is less than at the higher (alkaline) pH values, which is favourable, as more drug is needed in the initial stages of wound healing when the pH is alkaline and is susceptible to infection. Then with the passage of time and progression of wound healing, the pH starts decreasing towards the normal acidic range of skin that is about 5.5. As for comparison between C/G and H/G composites, the former shows more compliant release, as in the case of H/G the drug release is also higher near the neutral pH that is, 7.4 which is not necessary. Moreover, in H/G, the drug is not released in first 5 hours, when the wound is more vulnerable susceptible to infection.

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Figure 3.

Average drug release of hydrogels. (a) Chitosan-Gelatin, (b) HEMA-Gelatin.

Whereas in C/G, the drug release is higher within initial hours at higher pH and gradually decreases later, uniformly. But one little shortcoming is that at pH 6 the drug release is higher between 10-15 h than at all other pH values.

Figure 4(a) and (b) indicates the average degradation of both C/G and H/G samples at different pH. In case of C/G, the degradation was comparatively uniform. Its gel samples degraded rapidly at alkaline pH; within 12 h, but at acidic pH they kept swelling up to 48 h. The response of C/G at pH 7.4 was kind of neutral, in between acidic, and alkaline pH; it degraded after 24 h.

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Figure 4.

Average degradation of hydrogels. (a) Chitosan-Gelatin, (b) HEMA-Gelatin; transfer functions of best fit curve functions.

The hydrogel samples of H/G on the other hand showed stable behaviour at acidic pH. They neither swell to a high degree nor do they degrade rapidly. They survived till 48 h after keeping almost constant weight for a long time. As for the gels at pH 8 and 10, the weight kept increasing for 12 and 24 h respectively and then dropped quickly. The behaviour of samples at pH 7.4 was also a blend of acidic and alkaline pH, they absorbed plenty of solution, similar to samples at alkaline pH, but also remained constant like the samples at acidic pH after their degradation at 48 h.

Overall, the samples of both hydrogels at alkaline pH became shrunk and small whereas remained swollen in acidic pH till their degradation. They were swollen but friable and weak at neutral pH.

Anti-bacterial study results

Anti-bacterial study was also done in triplicates, the average diameter of zones of inhibition formed near drug loaded samples and positive control groups of both hydrogels, for three bacteria; E. coli, P. aeruginosa and MRSA are shown in Figure 5. The individual values for zone inhibition diameters are displayed in Figure 4 b along with the comparison of antibacterial activity for all sample types.

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Figure 5.

(a) Average diameter of inhibition zones formed by bacitracin zinc in E. coli, P. aeruginosa and MRSA culture (pictorial representation) (b) zone diameters for all control and experimental groups.

The results clearly display that both C/G and H/G have better antibacterial activity than positive controls in all three bacterial cultures when the amount of drug was kept equal in all. Between the two hydrogels, H/G depict better results than C/G.

The antibacterial assay must be correlated with both the drug delivery profile as well as the degradation profile. Drug Delivery profile shows a peak drug delivery just at 48 h. However, the rate of drug release starts increasing between 12 and 24 h. It is apparent from the results that the hydrogels undergo considerable swelling after 24 h to 48 time period. This indicates that composite samples absorb a copious amount of the surrounding moisture at this time. It is possible that this would be detrimental to the wound condition, and therefore, the cutaneous testing has been designed for 24 h, and microbial effectiveness is also checked after 24 h. This is rationalized further, as all the samples, positive, negative controls are treated in the same manner after each 24 h time period.

An alternative method may be explored with a detailed anti-microbial study in correlation with a detailed in-vivo study and this may be suggested as future works for this research.

SEM results

Surface analysis of both hydrogels show homogenous, compact and continuous structures (Figure 6(a)). The C/G surface is much smoother than H/G (Figure 6(b)) surface, which is because of the little particles of the initiators ⁵² used for the formation of H/G hydrogel.

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Figure 6.

a) SEM images showing surface morphology of hydrogels: Chitosan-Gelatin and HEMA-Gelatin; b) SEM images showing cross-sections of hydrogels. (a) Chitosan-Gelatin, (b) HEMA-Gelatin.

The cross-section areas of both hydrogels were also analysed through Scanning Electron Microscopy (Figures 6(c) and (d)). Generally, both the cross-sections have homogeneity, but again C/G’s cross-section is shown to have more structure than H/G.

FTIR results

FTIR result of C/G hydrogel without drug (Figure 7(a)) depict the Amide I and Amide II (Chitosan & Gelatin), Amide III (Chitosan) functional groups, N-H group bending vibration and imine bond (C-N) indicating the formation of covalent linkage between Gelatin and Chitosan through the Glutaraldehyde cross linker moiety. The only difference in the FTIR result of C/G hydrogel with drug (Figure 7(c)) is the increase in intensity of band at 1696 cm⁻¹ shows Bacitracin zinc binding to the gel matrix.

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Figure 7.

(a) ─ FTIR Spectrum of C/G hydrogel without drug. And FTIR Spectrum of C/G hydrogel with drug; (b) ─ FTIR Spectrum of H/G hydrogel without drug and FTIR Spectrum of H/G hydrogel with drug.

In the FTIR result of H/G hydrogel without drug (Figure 7(b)), stretching vibration for the OH group can be identified at 3600 cm⁻¹. A peak at 1014 cm⁻¹ may be attributed to the C-O-C group and the addition of Gelatin in the composite hydrogel may be identified by the 1762 cm⁻¹ peak. Increase in the intensity of band at 1600-1700 cm⁻¹ shows Bacitracin zinc binding to the gel matrix in the FTIR result of H/G hydrogel with drug.

Tensile testing results

The difference in the ultimate tensile strength of both hydrogels in dry and wet forms are depicted in Figure 8. The results show that C/G dry has the maximum Ultimate tensile strength of 38.21, and in wet form it is much decreased to 0.0321. Whereas, the ultimate tensile strength of dry H/G is much less than dry C/G, being 1.619 and even lesser in wet form which is 0.0179.

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Figure 8.

Mechanical tensile testing results of hydrogel samples in wet and dry forms. (a) C/G dry, (b) H/G dry, (c) C/G wet, (d) H/G wet.

Cutaneous application tests for the hydrogel systems results

The average results of wound diameter are described pictorially (Figures 9(a)–(d)) as well as in graphical form (Figures 9(a) and (b), Table 2) to compare the difference in healing.

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Figure 9.

(a to d) ─ Pictorial representation of wound healing in control and experimental groups; average change in diameter of wounds in each group during evaluation for 5 days. Percentage diameter reduction for all experimental and control groups in the cutaneous applicability test model has been represented.

The rate of healing for the excised wounds on the murine model’s dorsal side was recorded through perpendicular measurements at similar time intervals after a 24 hourly gap. The total time span of the recorded measurements was 5 days.

The initial wound diameter was 10 mm in all groups, which gradually decreased after healing. In negative control group the wound healing was very slow; there was negligible change in the healing rate within 5 days (that is, the wound closure progressed by only 2 mm, in the total time span that the wound healing was monitored). As for positive control group, the commercially renowned ointment Polyfax was applied daily, however the wound healed at a slower pace than experimental groups. Between the two experimental groups, results varied overall but in the final outcome (day 5), C/G showed slightly better results than H/G. The final diameter of C/G was 3.33 mm whereas in H/G it was 4 mm. The full scale of wound healing and closure is higher overall, for all experimental groups, as compared to both the positive and negative controls.

The in vivo measurements indicate that the healing in the untreated group is extremely slow. In both the experimental groups, the rate of healing is comparatively faster. Wound closure took place at a faster rate comparatively with in the positive control group, (with the same active ingredient ointment that was loaded into the hydrogels). Wound closure is notably accelerated in both C/G and H/G experimental groups. H/G hydrogel membranes performed nominally better with reference to wound closure, as compared to the C/G membranes.

Equilibrium water capacity

Equilibrium water capacity results are graphically represented in Figures 1(a) and (b). A difference in the degradation times. The occurrence of amine groups (-NH3) in both C/G and H/G gels mean that these systems are intrinsically, highly hydrophilic. The C-O-C linkage confers mechanical integrity to the individual hydrogel components. Even after swelling has occurred, the physical integrity of the hydrogels may be attributed to this linkage, facilitated by the Glutaraldehyde component.

It is also obvious from the equilibrium water capacity tests that for the same immersed volume of hydrogel samples, the EWC of HEMA/Gelatin samples is much lower as compared to Chitosan/Gelatin composite hydrogels. This could be attributed to the presence of the hydrophobic methacrylate groups in the HEMA backbone.

The EWC tests reveal information about the maximum potential of drug loading that these hydrogel systems can carry. Permeation based drug loading methods have been used to synthesize drug loaded hydrogel systems hence the quantity of dissolved solute absorbed, would ultimately reflect the final drug load capacity through immersion into a saturated drug solution. Hence it was feasible to carry out further experimentation on the hydrogel composite that indicates the highest EWC along with the most delayed degradation time. This high drug loading capacity would confer the hydrogel systems with a high drug dose (according to the requirement), a long degradation time, that could translate into extended usability in the clinical scenario. The C/G 1:3 composite and the H/G 1:4 composite were therefore tested further for drug loading and release testing. These hydrogel systems were also characterized further through spectroscopic analysis.

Degradation and drug release kinetics

The degradation and drug release kinetic studies are graphically represented in Figures 2 and 3. C/G at pH 4 indicates a biphasic pattern for drug release and a continual rate of swelling for up to 48 h, without any observable degradation. There is a probability that the swelling causes an influx of aqueous medium into the cross linked system and facilitates the subsequent release of drug dissolution and release. The pattern of drug release is vastly different from the H/G hydrogel system at pH 4, initially for 8 h of the study. H/G samples have a much higher swelling rate as compared to C/G, during these time slots. Drug release is also comparatively faster, for the first 18 h time span of the study, after which time point, there is an observable drop in the release rate. The decrease in the rate of release might be attributable to a drop in the amount of residual drug left in the system, since the drug release is highly dependent upon diffusion mechanisms (sink conditions being a primary driver for these mechanisms).Similar patterns for greater degree of swelling and drug release are observed for H/G for pH 6.

At pH 8 however, a reversal of events takes place. There is decrease in the rate of swelling of C/G hydrogels, however, simultaneously, an increase of the drug release rate is seen. This may be due to a shrinkage in the overall volume of the hydrogels (which may be correlated and traced back through gravimetric values, Figure 2). The shrinkage, that is chartable through depicted results, may cause the hydrogels to release any dissolved, but previously entrapped pockets of drug molecules, through a ‘spurting’ mechanism that is a consequence of the dramatic volume shrinkage in the hydrogel.

Similar trends are observable for both swelling, as well as drug release patterns with reference to H/G. Observed swelling and gravimetric increases might be because of protonation effects, of the amide groups that are present in C/G and H/G hydrogels. Repulsion of charges between the protonated functional groups can cause a lengthening of the chains that ultimately causes the swelling of hydrogels. The heavy prevalence of hydrophilic functionality in C/G and H/G hydrogels also causes copious amounts of aqueous medium to be taken up by the cross-linked systems.

The opposite pattern is observed for the alkaline pH segment, this is where deprotonation of the amine functional groups occurs, along with a shrinkage of the hydrogels. The volume decrease is reflected through both the drug release measurements as well as the gravimetric values. This observation could be clinically significant, since it may imply that a greater degree of drug might diffuse out of the hydrogel systems if both C/G and H/G systems are exposed to an alkaline pH milieu. This is usually the case in chronic or festering infected wounds. Wound pH gains greater traction towards the acidic end of the spectrum as it progresses towards the healing stage. This may also be correlated (through the current hydrogel based results), to a lower rate of drug release for the hydrogels. The synthesized hydrogel systems thus have potential to be useful in the clinical scenario, where wound physiology mediated release of medicament is required

Scanning electron microscopy

SEM results are shown in Figures 5 and 6. It is observed that the C/G hydrogels have a densely packed hybrid structure, which is covalently bonded, as indicated by the FTIR spectrum. Ultimately, this corresponds to a high degree of hydrophilicity; this aspect is also reflected through the EWC studies. There is slight stratification in the C/G composites.

For the H/G composites, there is a higher degree of homogeneity that corresponds to reasonably good pH sensitivity as observed through the drug release profile. A homogenous structure also corresponds to better interpenetration of the polymeric chains, consequently, we can also envisage that there will be less batch variability in this category of hydrogel composites.

Fourier transform infrared spectroscopy

FTIR results are depicted in Figures 7(a) and (b) and Figures 8(a) and (b). The FTIR spectra of the drug loaded samples depicts peaks for –NH₃, -C-H as well as –COOH groups (present in both categories of the hydrogels). The presence of the amine and carboxylic functional groups contribute greatly to the hydrophilic character of the hydrogel systems. To further confirm the incorporation of the drug in the matrix of the composite hydrogels, antimicrobial investigations have been carried out. The impregnation of the drug into the hydrogel matrix has been further established through UV based drug release studies in the ensuing discussion.

The presence of amine groups impart a highly hydrophilic and pH sensitive character to the hydrogel systems. Amine functional groups are protonated in the acidic milieu; this protonation is reversed in the basic extreme of the pH spectrum. This particular characteristic of the pH sensitive hydrogels has been extensively studied previously. ⁵³ The forces of repulsion between similarly charged, protonated functional groups, leads to a lengthening of the chains in the hydrogels; this phenomenon, coupled with the hydrogels intrinsic hydrophilicity, can result in the volume based swelling and inflow of external aqueous medium to the inner matrix. Consequently, a reversal of the surrounding pH and a dominance of the OH- groups, can lead to deprotonation and a shrinkage of the hydrogel volumes. This responses are reflected in the drug release patterns of both C/G and H/G hydrogel systems. The pH based stimulus responsiveness is imparted to the hydrogels through the presence of these functional groups and it is this intrinsic characteristic that has been harnessed in this research work for control and responsiveness with reference to drug release.

Cutaneous application tests for the hydrogel membranes

The results for cutaneous application tests indicate that the wound diameter reduction is quantitatively higher in the experimental groups (utilizing hydrogel membranes), as compared to the traditional topical antibiotic/dry and uncovered murine groups. This is also consistent with prior research consolidated by Junker et al. ⁵⁴ in the field of moist wound healing. It is established that wet or moist treatment of topical wounds leads to a reduction in the overall re-epithelialization time, and also reduces inflammation and necrosis in wounds. In a majority of studies, there has been no evidence of harmful or adverse reactions because of moist dressings, during the wound treatment phase. It is also understood that an environment which is moist can lead to the effective delivery of medication/antibiotic agents to the site of the wound. The research findings in the current cutaneous application study in the murine model are conducive to the fact, that the hydrogel systems accelerated wound diameter reduction and caused no harmful or adverse effects on a macroscopic scale. Further histological evaluation as detailed by Ansell et al. ⁵⁵ will be required in future studies, to fully evaluate the in-vivo effects of the developed hydrogel dressings.

Conclusion and future directions

The Equilibrium water capacity studies shed significant light on the absorption potential and hydrophilicity of the composite gels, thereby the drug loading capacity of the two hydrogel systems. This is further confirmed in the IR spectroscopic investigation, wherein the presence of a number of uncompromised (in case of the cross linked hydrogel systems) hydrophilic functional groups, impart the C/G hydrogels with a higher degree of hydrophilicity as compared to the H/G systems. Drug release and degradation studies that are carried out in tandem, broadcast the pH based biphasic nature of drug release for both categories of hydrogels. Charting out of the pH responsive drug release kinetics opens an insightful window to the potential of such hydrogels for use in the clinical setting for wound healing, where the pH of wounds is known to morph over an alkaline acidic spectrum over the complete time span of healing. The preliminary cutaneous application tests indicate that the hydrogels indicate that the hydrogels are physiologically triggered drug delivery systems; upon further extensive in-vivo testing, the clinical potential of these composite systems may be established. Our research data depicts that wound diameter reduction is three to five times quicker as compared to the negative groups, therefore, hydrogels showed promising results in this regard. On a macro-scale, no adverse effects of the dressings were noticed on the pace of wound healing; however, further evidence of the physiological effects may be established through histological evidence in future studies. Future investigations on the pH mediated drug delivery in infected wounds and ensuing biocompatibility of the hydrogels could also be carried out.

From the current drug release study experiments, it has been established that polymers-based dressings in contrast to hydrocolloids, facilitate and support bacitracin release effectively. No debris from the dressing was left in the wound, eliminating the need for extensive cleaning between dressings. Further to the results of the cutaneous application tests for the hydrogel systems depicted in this research work, some of the future works to be carried out should include detailed assessment of the current developed drug delivery system in with reference to stability data, the effects on stability, of any sterilization techniques used on the hydrogel based systems, as well as extensive in-vivo and subsequent clinical studies for validation of the results seen through the current experiments. Provided the biologically attuned hydrogels undergo successful clinical validation, these systems may be used in the hospital setting as well.