Formulation and evaluation of nano-ethogel of silver sulfadiazine for treatment of topical burns

Silver sulfadiazine (SSD)

Silver Sulfadiazine is a sulfonamide-based topical agent with broad spectrum antibacterial and antifungal activity. The chemical category of silver sulfadiazine is sulfonamides.

Silver sulfadiazine (SSD) is an efficient antibacterial agent approved by the USFDA, which is taken into account as the gold standard treatment in burn injuries.8, 9, 10

Mechanism of action of SSD8, 9, 10

Silver is a biocide, which binds to a broad range of targets. Silver ions bind to nucleophilic amino acids, further sulfhydryl, amino, imidazole, phosphate, and carboxyl groups in proteins, causing protein denaturation and enzyme inhibition. When SSD agent interacts with sodium chloride-containing body fluids, silver ions are released sluggishly and sustainably into wounded areas. Ionized silver atoms catalyze the formation of disulfide bonds resulting in protein structural changes and inactivating thiol-containing enzymes; silver ions may also fitDNA thereby snooping with replication and transcription of bacteria. As a competitive inhibitor of para-aminobenzoic acid (PABA), sulfadiazine inhibits bacterial dihydropteroate synthase, thereby acting in disruption of folic acid metabolism and eventually DNA synthesis.

Pathophysiology of burns

Burn is defined as an injury causing destruction to the epidermal, dermal or other deeper tissues on exposure to thermal, chemical or electrical agents. 11

A survey conducted by World Health Organization indicates that in a year approximately 1,80,000 people die due to burns. Therefore, it has been declared as a global public health problem by WHO.12, 13 According to the data of American Burn Association (ABA), there are over 4,50,000 of fatal burn injuries occurring alone in United States.14

Chemical sequences occurring at the time of burn

As a result of burns, proteins in the cells denature and coagulate. The burnt tissues stimulate the release of certain chemicals like histamine, kinin, serotonin and prostaglandin. Leukocytes and platelets adhere to the endothelium and cytotoxic T cells increase, therefore, making the tissue available as an open site for infection.^{_{11, 15}}

Injury due to burn occurs in two stages11, 16

The depth of injury is determined by amount of time period and degree of heat to which it is exposed.

Melting point determination

The melting point of the sample is done to check the purity of the sample. Melting point is defined as the temperature at which a solid substance transits its state from solid to liquid.17 Melting point of silver sulfadiazine was found by using the digital melting point apparatus from Remi Instruments.

Solubility analysis

Solubility is defined as the ability of a solute to dissolve in a liquid (solvent) to form a homogeneous solution. Factors affecting solubility are; type of solvent used, temperature and pressure.18

Solubility analysis was primarily performed in order to find out a suitable sol­vent to dissolve the API, lipid and excipients used for formulation preparation.

Partition coefficient of the drug

Partition coefficient is the measure of the lipophilic and hydrophilic nature of a drug substance. It is defined as the extent to which a substance is distributed between two liquid phases, one being the aqueous phase and other being the oily phase. The majorly used phases are water and n-octanol (oil phase) in the ratio 1:1.19

Determination of λ_max of silver sulfadiazine in 0.05% ammonia solution

Accurately weighed 100mg of silver sulfadiazine was added to a 100 ml volumetric flask. The drug was then shaken using 0.05% ammonia solution and volume was made up to the mark to get a stock solution. From this stock solution, aliquots of 100µg/ml were prepared by further dissolving 10ml of the above solution in 100ml of 0.05% of ammonia solution. The dilution was scanned from 400–200 nm with UV spectrophotometer keeping the 0.05% ammonia solution as blank. The spectrum of absorbance versus wavelength was recorded on UV-spectroscopy and analyzed for absorbance maximum and the highest absorbance was noted.20

Calibration curve of silver sulfadiazine using 0.05% ammonia solution

A 100µg/ml solution of silver sulfadiazine was prepared using 0.05% ammonia solution. This solution of 100µg/ml was used to prepare aliquots of varying concentration viz 2-18 μg/ml respectively. Absorbance of each of the solution was measured at 254nm in UV-spectrophotometer using 0.05% ammonia solution as blank and graph of concentration versus absorbance was plotted. The slope, straight line equation and correlation coefficient were obtained from the calibration curve intercept.

Preparation of silver sulfadiazine loaded ethosomal suspension

Ethosomal suspension of silver sulfadiazine was prepared based on the hot method suggested by Elka Touitou for the exploitation of ethosomal nanocarriers.

A dilution of silver sulfadiazine was prepared in 25% ammonia solution. Six different formulations i.e. F1, F2, F3, F4, F5 and F* were prepared by changing the concentration of ethanol and propylene glycol, along with the required amount of drug (500µg) by heating the preparation to 40˚C followed by mechanical stirring at 400rpm. Lecithin was suspended in required quantity of water for a few hours and was quickly stirred mechanically at 900-1100 rpm and then heated to 40˚C so that it was completely bloomed at the time of usage. Lipid phase was added dropwise to ethanol mixture with continuous and closed magnetic stirring at room temperature for 30 min at about 300 rpm to achieve uniform mixing. The formulation was finally sonicated using a probe sonicator (Frontline Sonicator) for about 5-8 min keeping in an ice bath (4˚C). A homogenous SSD ethosomal suspension was obtained.21 The nano-ethosomal formulation was stored in a covered vessel at room temperature, away from light for further experimentation.

Composition of SSD ethosomal formulations is given in Table 2 below.

Preparation of SSD Loaded Ethosomal Gel (Ethogels)

Hydrogels were prepared by dispersing 1.5% w/w of Carbopol 934 in distilled water. The Carbopol hydrogel was neutralized by addition of triethanolamine dropwise, and then the ethosomal suspension in a ratio of 1:1 (1 part of suspension: 1 part of gel) was added to it at 200 rpm for 5 min to get a homogeneous dispersion.

Figure 2

Silver sulfadiazine loaded ethosomal formulations

Figure 3

SSD-loaded ethogels

Evaluation of prepared SSD-loaded ethogels

Content uniformity and drug content

The drug content of the prepared gels were conducted by dissolving accurately weighed quantity of 1g gel in 100 ml volumetric flask and volume was made up to 100 ml with 0.05% ammonia as a solvent. The contents were magnetically stirred for 2-3 hours. The contents were filtered followed by making appropriate dilutions. The prepared dilutions were analyzed spectrophotometrically using an UV spectrophotometer at a maximum wavelength of 254nm.

In vitro drug release

The in vitro drug release studies from prepared SSD ethosomes were performed using dialysis bag diffusion technique.

0.5ml of each prepared SSD ethosomal formulation was filled into the diffusion bag and bag was precisely tied with threads at both the ends and suspended in a beaker which was filled with phosphate buffer solution (PBS) at pH 7.4. The prepared model was stirred with the help of a magnetic stirrer (Remi Equipments, India) at 50 rpm. Samples were withdrawn from the receiver medium at time intervals of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 hours and then were replaced with equal volumes of fresh PBS. The pH was mandatorily maintained to 7.4 every time. The amount of drug released in buffer was determined using a UV-Vis spectrophotometer at 254 nm, keeping PBS as blank.

Stability study

Ethosomal gel formulations were stored in well closed glass beakers under the following conditions and testing was performed at the specified time points:

Room temperature at 25±˚C: analysis was performed after the 14^th and 28^th days of storage.

Refrigerated temperature at 4±2˚C: analysis was done after the 14^th and 28^th days of storage.

Identification of pure drug

Particle size of pure SSD

Particle size was determined using Malvern Zetasizer and was found that all the particles have an average size range lying between 0.1 to 12 μm. The graph obtained is depicted in Figure 4.

Figure 4

Particlesize distribution curve of pure silver sulfadiazine

Standard calibration curve of silver sulfadiazine

The calibration curve of Silver Sulfadiazine was obtained by using the 2-18µg/ml concentration of Silver Sulfadiazine in 0.05% ammonia solution. The absorbance was measured at 254nm. The calibration curve which was plotted was linear in the range of 2-18μg/ ml at λ_max 254nm. The correlation coefficient was found to be 0.996. The calibration curve is depicted in Table 3 and Figure 5.

Table 3

Silver sulfadiazine calibration curve (λ_max = 254 nm)

S. No.	Concentration (μg/ml)	Absorbance
1.	2	0.136
2.	4	0.19
3.	6	0.29
4.	8	0.393
5.	10	0.479
6.	12	0.556
7.	14	0.622
8.	16	0.718
9.	18	0.785

Figure 5

Calibration curve for silver sulfadiazine

Table 4

Result of regression analysis of UV Method

Statistical Parameters	Results
λ max	254nm
Regression equation (y = mx + c)	0.041x + 0.046
Slope (m)	0.041
Intercept (c)	0.046
Correlation coefficient (R2)	0.996

Table 5

Physicalappearance, ph, viscosity, spreadability and extrudability of the prepared gel

Formulation	Appearance	pH	Viscosity (mPa.S)	Extrudability (gm)	Spreadability (gm.cm/sec)
F1	Light brown ,Homogenous	6.7	24,000	0.5	7.67
F2	Mud brown, Homogenous	7.0	26,000	0.5	7.63
F3	Mud brown, Homogenous	6.8	38,000	1.3	6.84
F4	Light brown, Homogenous	6.4	34,000	1.8	10.11
F5	Light brown, Homogenous	6.7	43,000	1.6	14.13
F*	Light brown, Homogenous	7.2	29,000	2.2	10.30

Figure 6

pH, extrudability and spreadability of the prepared gel

Table 6

Percentage drug content of SSD loaded topical ethogels

S. No.	Formulation Code	Drug Content (%)
1.	F1	46.44
2.	F2	48.24
3.	F3	53.30
4.	F4	81.64
5.	F5	74.59
6.	F*	70.34

Table 7

In Vitro drug release (%) of different formulations of SSD ethosomes

Time (hr)	F1	F2	F3	F4	F5	F*
0	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
0.5	6.14±0.92	8.90±0.010	11.93±0.041	14.55±0.204	16.20±0.05	16.18±0.096
1	8.70±0.39	13.64±0.05	15.80±0.96	26.54±0.13	28.53±0.154	22.23±0.041
1.5	11.45±0.285	20.71±0.204	23.23±0.041	34.19±0.53	31.70±.067	27.61±0.204
2	16.92±0.35	28.19±0.72	29.35±0.05	44.65±0.267	36.75±0.020	32.04±0.13
2.5	23.83±0.041	31.70±.067	35.44±0.228	52.14±0.92	40.95±1.02	38.10±0.678
3	27.61±0.204	36.75±0.020	41.74±0.03	60.72±0.336	48.52±0.86	44.65±0.267
3.5	32.04±0.13	40.95±1.02	45.35±0.04	65.84±0.204	56.98±0.852	51.14±0.72
4	37.80±0.204	46.13±0.327	52.14±0.92	69.43±0.92	60.72±0.336	57.52±0.67
5	48.23±0.285	53.52±0.231	60.72±0.336	76.80±0.204	71.54±0.204	65.84±0.204

Figure 7

In Vitro drug release (%) of different formulations of SSD ethosomes

Table 8

Stability studies of SSD loaded nano-ethogel

2 Weeks	Parameters	F1	F2	F3	F4	F5	F*
	Appearance	Light Brown	Muddy Brown	Muddy Brown	Light Brown	Light Brown	Light Brown
	pH	6.7	7.0	6.8	6.4	6.7	7.2
	Viscosity	24,000	26,000	38,000	34,000	43,000	29,000
	Spreadability	7.67	7.63	6.84	10.11	14.13	10.3
	Extrudability	0.5	0.5	1.3	1.8	1.6	2.2
	% Drug content	46.44	48.2	53.3	81.64	74.59	70.34
1 Month	Appearance	Light Brown	Greenish Brown	Greenish Brown	Light Brown	Light Brown	Light Brown
	pH	6.7	9.2	8.8	6.4	6.7	7.2
	Viscosity	24,000	26,000	38,000	34,000	43,000	29,000
	% Drug content	46.44	44.90	51.6	81.64	74.59	70.34

Evaluation of prepared SSD-loaded ethogels