Microsphere Loaded Silver Compound for the Effective Management of Burn

INTRODUCTION

Burns are injuries caused by heat, friction electricity radiation/ chemical to the body tissue. It may cause swelling, blistering, scaring, and shock and in severe condition even death.¹ Serious thermal injuries occupy a large percentage of body area and require immediate care, management, and treatment to minimize the possible mortality and morbidity. Wound healing is a complex process in which wound gets contracted and closed along with restoration of the functional barrier. Even though antimicrobial chemotherapy and management are effective for wound healing, infection still remains a big problem. During infections, bacteria stay intracellularly and impair antibiotic treatment and delayed natural healing process. Hence a more effective, and cost efficient treatment strategy is required.^2,3

Particulate drug delivery systems (DDS) when applied to open wounds provide water vapour and oxygen permeability to wounds, increase bioadhesiveness and also release the drug in a controlled manner which in turn speeds up the healing process.⁴ SSD containing silver having antimicrobial property controls yeast, mould, fungi, and bacteria had been used anciently. SSD ionize to Ag ions which disrupt microbial DNA, interfere with metabolic process like DNA synthesis, folic acid pathway, and interact with thiol group in microbial proteins.⁵

Conventional therapy requires frequent application and may produce delayed healing and systemic toxicity. Hence novel DDS has to be designed to modify and pharmacokinetically control the drug release. To over come all these issues a specialized drug delivery system that localize and deliver the antimicrobials in a controlled manner is to be usedto prevent antimicrobial infection and allow natural healing. Particulate systems like liposomes and microspheres have been investigated for delivery of drug to specific skin compartment in order of drug release, input, retention and action into the skin and decrease in systemic absorption and subsequent ADR.⁵ This study is an attempt to formulate a microsphere loaded cream of SSD for controlled drug delivery to burns thereby reducing the limitations of conventional therapy.

MATERIALS AND METHODS

Preformulation studies

Preformulation studies were an investigation of physical and chemical properties of a drug substance. FT-IR studies were performed to confirm the compatibilityof drug and excipients in the formulation. FT-IR of pure drug individually and in combination was taken and was compared to the reference spectrum. The preformulation studies also included the determination of melting point (MP) using capillary tube method and loss on drying (LOD) as per USP methods.

Preparation of microspheres^6,7,8

Formulation containing different polymers, chitosan and sodium alginate, at concentrations of 2, 3 and 4%w/v, in different drugs to polymer ratios of 1:2, 1:3, and 1:4 were used for the formulation development. Drug polymer dispersion was prepared by dispersing the drug into the polymer solution which was prepared by dissolving polymer in 2%v/v acetic acid at room temperature with vigorous stirring. W/O emulsion was prepared by adding the dispersed phase into the continuous phase of light liquid paraffin containing 0.5% w/v of Span 80. Continuous stirring of the above content at 2000 rpm was done using stirrer. After 30 m of homogenization glutaraldehyde saturated toluene (GST) was added at time intervals of 15, 30, 45 and 60 m15,30,45. The above mixture was kept aside for cross linking for a period of 5 h and finally the microspheres were separated by decantation and filtration.

Evaluation of microspheres^9,10

Particle size determination: Average particle size of microspheres was measured using optical microscopy. Particle size determined using the following equation

D mean = ∑n.d / ∑n

%Entrapment efficiency (%EE) and % yield: The microsphere were weighed and dissolved in acetic acid and made upto 100 mL with PBS. The 

% Drug loading= amount of drug microsphere / amount of microsphere 𝑋100

% EE= amount of drug loaded / initial amount of drug 𝑋100

% Yield = wt. of microsphere / wt. of solid starting material 𝑋 100

absorbance was measured at 263 nm using UV spectrophotometer (UV1700, Shimadzu, Japan).

In vitro drug release: Drug loaded microspheres equivalent to 100 mg of drug was introduced to a basket type dissolution apparatus and dissolution was carried out using standard procedure at 100 rpm and aliquots of samples withdrawn at various time intervals for a period of 24 h. The sink condition was maintained throughout the study.

Preparation of SSD loaded microspheres cream^11,12,13: Oil soluble ingredients were dissolved in oil phase and water soluble ingredients dissolved in aqueous phase separately at 70°C. The cream base was then prepared slowly adding oil phase into aqueous phase with vigorous stirring. A specified quantity of SSD loaded microspheres equivalent to 1g from selected formulation was incorporated into the cream base by simple mixing.

Evaluation of SSD loaded microsphere cream¹⁴

Spreadability: Determined using a specially designedspreadability apparatus. Spreadability

S = M.L / T

M = weight tied to upper slide, L= length of glass slide, T = time taken to separate slides

Rheological study: Viscosity of the formulation was determined using Brookfield viscometer LV DV prime 1 at different rpm.

Drug content: Weighed amount of cream was dissolved in suitable solvent and absorbance was measured at 263 nm using UV spectrophotometer (UV 1700, Shimadzu, Japan).

In vitro diffusion study: Receptor compartment of Franz diffusion cell contained PBS of pH5.8. Specified amount of cream was applied evenly on the cellophane membrane and it was placed between the donor and the receptor compartments. Aliquots of sample was collected at various time intervals and analyzed at 263 nm using UV. The cumulative amount of drug release across membrane was found out as a function of time.

In vitro antimicrobial study: The bacterial strains (Pseudomonas aeruginosa) were dispersed into the sterile medium and the medium was poured into sterile petridishes which was cooled and solidified. Wells of 6mm was made in the medium and transferred with microsphere loaded cream formulation. The procedure was repeated with the cream base and marketed SSD cream. The plates were incubated for 37°C for 24 h in an incubator. The zone of inhibition was observed and measured.

Stability studies¹⁵

The formulated SSD microsphere preparation was packed in suitable borosilicate container and stored at 40 ±2°C, 75±5% RH of 45 days. The formulation was then evaluated for viscosity, spreadability,and drug content.

RESULTS

Preformulation

The MP was found to be 285 ±0.56 ºC and the LOD of pure sample was 0.5%. The FT-IR spectrum of the components of the formulations was found to be similar to the reference spectrums. The results were found to be satisfactory and within the range.

Preparation and evaluation of microsphere: The microsphere were prepared using the method explained previously and the evaluation studies for % entrapment, %yield, particle size, and in- vitro drug release for all formulation were carried out.

Preparation and evaluation of microsphere loaded cream: The prepared SSD microspheres were incorporated into the cream base, and evaluation studies were carried out. The best formulations FC3 & FS3 were evaluated for their viscosity, % drug content, spreadability, in vitro drug diffusion, and antimicrobial study.

DISCUSSION

Preformulation studies

Studies suggested no incompatibility between the drug and the excipients.

Preparation of microspheres

Microspheres are prepared by emulsion cross linking solvent evaporation method. Six formulationsof SSD microspheres were prepared by keeping drug, stirring speed, volume of cross linking agent, stirring time, emulsifying agent, etc. constant and varying only the concentration of polymers. Formulation details were recorded in the Table. 2

Evaluation of prepared microspheres

Particle size: Size of prepared microspheres varied from 84.84±2.54µm to 100.19±3.78µm. When concentration of polymer increased the size also increased. Out of 6 formulations 4 %w/v chitosan SSD FC3 and 4%w/v sodium alginate FS3 were found to be optimal for topical application.¹⁶

%yield: All formulations showed good percentage yield. % yield of 4%w/v chitosan and 4%w/v sodium alginate FC3 and FS3 were 87.8±0.94, 86.01±0.51, respectively. % yield increased with increase in polymer concentration. When polymer to drug ratio increased yield also increased.⁴ Chitosan microspheres showed more yield than alginate microspheres.

%Entrapment efficiency (%EE): It ranged between 59.24±1.08, and 76.02±0.83%. Among the polymers used 4% w/v of both polymers showed good %EE, 76.02±0.83% (FC3), and 74.97±0.86% (FS3). It was found that, when polymer concentration was increased entrapment efficiency also increased.

In vitro dissolution study: In-vitro dissolution study showed that as the polymer concentration increased dissolution rate decreased. At the end of 24 h, 2% w/w concentration showed 81.39±0.15 %( FC1) and 78.51±0.62 %( FS1) and 4 %w/v concentration showed 75.76±0.90 % (FC3) and 75.31±0.58% (FS3) of drug release. From the results we can say that FC3 and FS3 showed slightly extended drug release compared to other formulations.¹⁶ Also these two formulations were found to be the optimized ones.

Preparation and evaluation of SSD loaded microsphere creams: Optimized microsphere formulations FC3 and FS3 were incorporated into the cream base. Spreadability of FC3 and FS3 were found to be 5.69±0.28 and 5.58±0.45, respectively. This shows good spreadability. The viscosity of the formulation showed a pseudo plastic behavior which is a desirable property for topical preparation.¹⁷ Drug content of FC3 and FS3 were found to be 92.4% and 88.9%, respectively, which show the ability of the base to incorporate the maximum quantity SSD microspheres. In case of antimicrobial study it was found that FC3 having 4% chitosan had maximum zone of inhibition of 21 mm when compared to FS3 containing 4% alginate which is having a zone of inhibition of 17mm.

In-vitro diffusion study: At 8 h FC3 and FS3 formulations showed 57.03±0.87%, 62.09±.68% release, respectively. Whereas marketed formulation had 95% release. From all the above evaluations, it was found that FC3 was superior to FS3 and it was used for stability studies.  

Stability studies

Accelerated stability studies as per ICH guidelines with little modification were performed. Stability test for FC3 formulation was done for 45 days and showed that the formulation passes the stability test.

CONCLUSION

To reduce the risk of systemic toxicity and infections after burn, microspheres can be an effective carrier for delivery of SSD topically. Hence in the study an attempt to develop SSD loaded microsphere cream has been successfully completed. The compatibility studies using FTIR showed no possible interaction between the excipients. From the study it is very clear that the pre and post formulation studies show satisfactory results. Out of 6 formulations FC3 (4% chitosan) and FS3 (4% sodium alginate) passed all the evaluations like particle size, entrapment efficiency, % yield, antimicrobial study, diffusion, and dissolution. In the subsequent studies FC3 i.e. chitosan loaded microsphere cream was found to be the best formulation which complies with even accelerated stability studies. Finally we can conclude that the study was a successful attempt to formulate and evaluate controlled release topical formulation of silver sulphadiazine in order to reduce the frequency of application, systemic toxicity, and infections.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.

ACKNOWLEDGEMENTS

I take this opportunity to express my thankfulness to my guide, and college authorities for facilitating in carrying out my research work.