INTRODUCTION:

Conventional topical cream formulations with antibiotics are unable to deliver the drug effectively over the bacterial affected wounds in the skin hence necessitate longer treatment duration or have to be supplemented by oral therapy. Mupirocin (MUP) is an antibiotic used to treat skin surface aerobic bacterial infections. It is effective against Staphylococcus and Streptococcus bacteria, and it is also utilized to control Methicillin-Resistant Staphylococcus aureus mediated infections.^1-3 During the past decade anaerobic bacteria have been established as a organisms of most of the infection including burn infection Tinidazole is nitro imidazole derivative, anti-parasitic drug against protozoal infection and gram negative anaerobic bacteria Bactriodes fragilis. Solid lipid nanoparticles (SLNs) were developed at the beginning of the 1990s as an alternative particulate delivery system to liposomes, emulsions, microparticles, and polymeric nanoparticulate systems. SLNs have drawn major attention as novel colloidal drug carriers for the administration of drugs by several routes such as oral, topical, parenteral, ophthalmic, and rectal.^4-6

MATERIALS AND METHODS:

A gift sample of Mupirocin and Tinidazole received as gift sample from (Fourtt’s laboratory Chennai). Glyceryl monosterate, Tween 80, Sodiumalginate, (Lobachemical Ltd., Mumbai) Stearicacid, (E.merckindia Ltd., Mumbai) pluronic F127, F68 (Himedia Laboratories, Mumbai) propylparaben (Fischer Scientific, Mumbai) were purchased.

Methods:

Hot-homgenization method:

Glyceryl monosterate and stearic acid were taken in required quantities and melted together at approximately 80°C. To this melted lipid mixture Mupirocin (600 mg) and Tinidazole (300 mg) were added simultaneously. To this 30 ml of phosphate buffer pH 7.4 containing surfactant 0.5% tween or polaxamer 188 was added at the same temperature 80°C. The emulsion obtained was stirred for 30 min using mechanical stirring and then it was subjected for high speed homogenization for 2.5 min at 5000 rpm. The emulsion was then sonicated using probe sonicator for 2 min which results in the formation of SLN⁷.

Tablet:1Formulation of solid-Lipid nanoparticles with Mupirocin and Tinidazole

Formulation	Stearic acid	Glyceryl monosterate	Tween 80	Poloxamer188
F1	1500 mg	-	0.5 ml	-
F2	1500mg	-	-	250 mg
F3	-	1500 mg	0.5 ml	-
F4	-	1500 mg	-	250 mg

Characterization of solid-lipid nanoparticles:

Particle size, Polydispersity Index and Zeta potential analysis:

The size analysis and Zeta potential, polydispersity index of the SLNs were determined using a Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Each sample was suitably diluted with filtered distilled water (up to 2 ml) to avoid multi-scattering phenomena and placed in a disposable sizing cuvette. The polydispersity index was studied to determine the narrowness of the particle size distribution. Zetapotential was ranged from -200 to + 200 Mv. The electrophoretic mobility was converted to zeta potential by in built software using the Helmholtz – Smoluchowski equation. The size analysis and zeta potential of a sample consisted of three measurements, and the results were expressed as mean size average⁸.

Atomic force microscopy :

The best formulation liquid sample was adsorbed on the surface of silicon wafer and allowed to dry at room temperature. The sample were examined using multimode scanning probe microscope (NTMOT, NTEGRA Prima, Russia) in semi conduct mode with a force constant range of 0.35-6.06N/M and a resonating range of 47-150 KHZ. The phase images was used to determine the morphology of Mupirocin-Tinidazole solid-lipid nanoparticles.

Entrapment efficiency:

The entrapment efficiency (EE), which corresponds to the percentage of mupirocin and Tinidazole encapsulated within and adsorbed on to the nanoparticles was determined by measuring the concentration of free mupirocin and tinidazole in the dispersion medium. A volume of 1.5 ml of drug-loaded sample was centrifuged at 10000 rpm for 20 min to separate the lipid and aqueous phase. The supernatant was then diluted with phosphate buffer pH 7.4and analyzed by UV VIS spectrophotometer at 223 nm and 282 nm⁹

Encapsulation efficiency was calculated as follows:

EE (%) = [(Ct-Cr)/Ct] X 100

Where,

Ct –Amount of drug added

Cr—Amount of free drug in supernatant

In - vitro release studies:

The In -vitro drug release of Mupirocin and Tinidazole from different SLNs dispersion without free drug separation, free drug separated nanoparticles and gel was determined by using dialysis bag diffusion technique. An accurately weighed 2 ml of SLNs was transferred to a dialysis bag and sealed. The sealed bag was then suspended in a beaker containing 250 ml of phosphate buffer pH7.4 and stirred at a constant speed of 100 rpm at 37°C ± 0.5°C. Aliquots were withdrawn sample at every one hour intervals from the receptor compartment upto 6 hours and the same replaced with fresh buffer. Then the drug content was determined spectrophotometrically by measuring the absorbance 223 nm for Mupirocin and 282 nm for Tinidazole using the respective receptor medium as a blank to calculate the amount of drug released from nanoparticles.^9-10

Stability studies:

Stability studies were carried out for the optimized formulation having high entrapment efficiency and In-vitro drug release by storing the formulation at two different temperature 4°C and 25± 2°C and the particle size was estimated at 15 days, 30 days and 45 days¹⁰.

SLNs loaded in Topical gel:

All the three polymers that is Pluronic F 127, Pluronic 68 & Sodium alginate were mixed in appropriate quantity of distilled water and stirred separately for 1 h on magnetic stirrer and refrigerated overnight at 4°C. Next day, Pluronic F 127 &Pluronic 68 polymer solutions were mixed together with continuous stirring for 1 h and again kept overnight at 4°C. Sodium alginate solution along with methyl paraben were added to above mixture and mixed with continuous stirring for 1 h. This mixture was added to specified quantity of SLN formulation F1 and F2 and sodium chloride solution in distilled water with continuous stirring until it dissolves and further it was adjusted to pH 7.4 by adding 0.1 N sodium hydroxide solution. The formed gel was stored in the refrigerator 2-8°C¹¹. Two formulations were prepared by varying the concentration of Pluronic 127 as mentioned below.

Table : 2 SLNs loaded in topical gel

Formulation ingredients	F1	F2
SLN eq. to. gel	25 ml	25 ml
Pluronic F 127	3.5 g	3.0 g
Pluronic F 68	0.75g	0.75 g
Sodium alginate	2 g	2 g
Methyl paraben	0.01g	0.0l g
Benzolkonium chloride	q.s	q.s
Distilled water %)	25ml	25 ml

Characterization of solid-lipid nanoparticles loaded in topical gels:

Organoleptic character:

All the drug loaded cream formulation were tested for physical appearance, color, texture, phase separation and homogeneity. These characteristics were evaluated by visual observation (Fedelic Ashish Toppo et al., (2016) Homogeneity and texture were tested by pressing a small quantity of the formulated cream between the thumb and index finger The consistency of the formulation and presence of coarse particles were used to evaluate the texture and homogeneity of the formulations.¹⁰

pH evaluation:

The pH of various formulation was determined by using digital pH meter. About 1g of the gel was weighed and dissolved in 25 ml distilled water and stored for two hours. The measurement of pH of each formulation was done in triplicate and average values were evaluated¹¹.

Viscosity:

Brookfield viscometer was used to measure the viscosity of gel. For this purpose, spindle No. 04 was used. Viscosity was recorded by rotating spindle at 10 rpm¹¹.

Anti-bacterial activity studies:

MIC (Minimum inhibitory concentration):

MIC determination were performed in 96- well microplate according to procedure described by the clinical and laboratory standards institute. Column 1-12 were filled with 100 µl of sterile Mueller broth. Concentration were in the ranges of mupirocin solid-lipid nanoparticles and SLNs based gel: 0.075 µg/ml - 50 µg/ml. Accurately 100µl of anti-microbial solution (mupirocin was added 1^st column 100 µl solution was taken from column 1and diluted two fold by transferring them into column 2 with a pipete.100µl solution was then removed from column 2 and transferred to column 3and so on through to column 10.The last 100ul of diluted drug is then discarded. Each well was inoculated with 5 µl bacterial suspension equal to 0.5 MC Farland standard turbidity. The growth control well (column 11) which contained 100ul of the same inoculum suspension. Column 12 filled 100µl of sterile media without any culture. The microplates were incubated at 36^o c for 24 hours under aerobic condition and the bacterial growth was confirmed by adding 10 µl of a sterile 0.02% aqueous solution of resazurin sodium (Hi-media labs)and then incubated at 36^o c for 4 hours. The viable bacterial cell change resazurin sodium blue colour to pink colour was noted¹².\

Zone of inhibition:

The 10µl of solid-lipid nanoparticles and SLNs based gel solution was added to separate sterile disc then it was dried. The dried disc was placed in petri dish Gentamycin disc (10µl) was used as positive control. Then it was inoculated with Staphylococcus aureus (ATCC No 23235) and Pseudomonas aeruginosa (ATCC No 15442). Petri plates were incubated at 37^oc for 24 h. The sensitivity of test organism to the formulation were indicated by clear zone of inhibition around the disc and the diameter of the zone of inhibition was measured¹³.

RESULTS AND DISCUSSION:

Particle size, polydispersity index and zeta potential:

Two different surfactant of Poloxamer 188 and Tween 80 were used. In this study for the formation of SLN. SLNs with Poloxamer 188 produced particles of 211 nm whereas Tween 80 produced particles with size of 83 nm. The PDI values ranged between 0.036 and 1.00, indicating that the produced nanoparticle populations in formulation F1and F2 have a good degree of homogeneity (Table 3). Zeta potential values in the range 15 to 30 mV are common for stabilized SLNs.

Thus, the zeta potential values indicate that the electrostatic repulsion between particles will prevent their aggregation and thereby stabilize the nanoparticulate dispersion¹⁴. In this study zeta potential values are in between (-2.1 to -5.2). Low zeta potential values are due to the presence of non-ionic surfactants in the formulation. Even though the charges are less aggregation of particles are not observed during storage. It confirms nonionic surfactants poloxamer 188 and tween 80 also able to provide steric stability to the SLNs (Table 3) Significant difference in particle size was observed among four formulation with P value (P<0.001)

Table : 3 Particle size, zeta potential and PDI

Formulation	Particle size (nm)	Zeta potential	PDI
F1	83.6	- 2.2 mv	0.036
F2	211.2	- 5.2 mv	0.306
F3	2228	-2.1 mv	0.487
F4	4830	-4.2 mv	1.000

Atomic Force Microscopy (AFM):

AFM results obtained showed that the particles were spherical in shap

Fig :1 AFM Images

Entrapment efficiency:

Entrapment was high in F1 formulation respective to mupirocin a tinidazole when stearic acid as a lipid and tween 80 used as surfactant was used. But entrapment was found to be less F2 Formulation when surfactant poloxamer 188 was included instead of Tween 80.Entrapment was less even glyceryl monosterate and tween 80 and poloxamer 188 was used in the formulation F3 and F4. There is significant difference in entrapment efficiency between all four formulation p<0.001. But no significant difference between F1and F4 formulation.

In vitro drug release:

In matrix systems such as lipid nanoparticles, the drug is incorporated in the lipid matrix either in dissolved or in dispersed form¹⁴. Comparative drug release studies were carried out for plain drug solution, SLNs dispersion, and SLNs based gel and results are shown in Fig 2, 3, 4.

Fig : 2 In-vitro drug release of mupirocin and tinidazole (without free drug separation)

Fig 3 : In vitro drug release of bound solid-lipid nanoparticle

SLNs dispersion showed initial burst release of 37.5% of mupirocin (SLNs) and 45% tinidazole from formulation F1 in 1hr (Fig 2). Significant drug release was observed for mupirocin from SLN at 1 hr between F1 vs F2, F1vs F4 formulation (P < 0.007). Significant drug release was observed for mupirocin from SLN at 6 hrs between F1 vs F3 (P < 0.01) Significant drug release (P< 0.01) was observed for tinidazole at 1 hr between F2, F3, F4 formulation. No significant F1vs F2 F1vs F3 and F1vsF4. Significant drug release was observed for tinidazole from SLN at 6 hrs between F1 vs F3 (P < 0.09)

But the solid-lipid nanoparticles containing only bound drug shows 20% mupirocin and 32.5% tinidazole drug release at 1 hour which was less compare to without free drug separation(Fig 3). Deposition of the drug in the outer layers of the particles resulting in superficial entrapment may be the reason for initial burst release. Mupirocin and Tinidazole entrapped deep within the nanoparticles sustains the release up to 6 h. The slow release in the later stage was attributed to the fact that the solubilized or dispersed drug can only be released slowly from the lipid matrices through dissolution and diffusion mechanisms.

Fig : 4 in - vitro drug release SLNs loaded in topical gel

In SLNs based gel F1 formulation 37.5 % of mupirocin and 32.5 % of tinidazole got released respectively in 1 h followed by sustained release up to 6 hrs (Fig 4). In Korsmeyer–Peppas equation, n is a diffusion exponent that characterizes the drug transport, n value for SLNs dispersion was 0.38 for F1 formulation indicating the fickian drug diffusion (n < 0.5).

Stability studies:

Formulations stored at both temperature conditions (room and refrigerator condition) remained in their colloidal Particle Size (PS) range. Initial PS of F1 formulation SLNs was 126 nm in room temperature and 89 nm in refrigerated condition. Upon storage, slight increase in particle size 142 nm was observed for formulations stored at room temperature as compared when stored under refrigerator condition 92 nm after 45 days which may be attributed to the aggregation of lipid particles. As a conclusive suggestion, however, storage in a refrigerator or anyhow at a controlled temperature below 25°C could be advised for these systems

Organoleptic characteristics:

Table:4 Organoleptic characteristics

F. code	Physical appearance	Color	Texture	Phase separation	Homogeneity	pH
F1	Opaque	White	Smooth	No	Homogenous	6.1
F2	Opaque	White	Smooth	No	Homogenous	6.2

Drug content in Mupirocin –Tinidazole loaded SLNs based gel F1 was found to be 78%. pH of SLNs based gel was found to be 6.1 for F1 formulation and 6.2 for F2 formulation which is acceptable for topical formulation as pH of normal skin is 5.5. After incorporating SLNs loaded mupriocin in the sodium alginate gel viscosity increased to 5100 cps from 4900 cps. The SLN dispersion with and without drug possesses low viscosity 4990 from 5100 and hence it is inconvenient for topical application. Gel preparations are normally preferred because of their controlled release character

In addition, aqueous dispersions of solid lipid nanoparticles are basically not stable for longer time and some systems show particle growth followed by gelation^{15, 16}. However, the nanoparticulate structure can be maintained, and aggregation and gelling phenomena of the particles could be avoided by incorporating the dispersion in gels. Hence, we incorporated the SLNs into sodium alginate

Spreadability:

Spreadability of prepared gel 399 g was found to be closer to that of standard 364 g mupirocin cream it confirms the prepared gel able to spread easily compared to cream

Extrudability:

Extrudability of prepared gel 1014 g was found to be closer to that of standard mupirocin cream 1027 g. It confirms the gel will able to come out container with less force like that of cream but the extrudability value for sodium alginate base alone was 927g

Bloom strength:

Bloom strength of the gel was less -0.231g when compare to cream. 5.78g. Bloom strength was low compared to cream. The strength of the gel with 2% sodium alginate gel was less -0.123g.

Anti- bacterial studies for SLNs and SLNs loaded in topical gel:

Experimental data demonstrated that the nanoparticles produced with nonionic surfactant Tween 80 able to produce (12 mm) of zone of inhibition, poloxamer 188 (10 mm) inhibition zone but free mupirocin antibiotic able to produce more zone of inhibition (18 mm) against the tested bacterial species S. aureus and P. aeruginosa. Such a behavior can be described to the lipid nature and low charge of these nanoparticles that have not allowed for good interaction with the bacteria surface and penetration through their cell wall. However, among the most recent published articles, SLN loaded with ciprofloxacin have been studied by plate diffusion testing against S. aureus and P. aeruginosa. From the published results, it was not possible to evaluate the inhibition zone, the authors however concluded that the nanoparticle formulation showed an higher antibacterial effect than the pure drug¹⁷.

The drug was dispersed within the lipid matrix which was further incorporated into sodium alginate bioadhesive gel which enhanced the diffusion of the drug into the media results in high zone of inhibition (22 mm). Inclusion of non-ionic surfactants used for stabilization of SLNs at low concentration may also influenced drug diffusion. The fact that the drug diffusion through media was more in case of SLNs based gel than pure drug (19 mm) confirming the formulation of SLNs based drug delivery system may be useful for skin targeting and controlling the drug release.

Fig : 5 Zone of inhibition of mupirocin SLN loaded sodium alginate topical gel formulation

Table : 5 Comparative zone of inhibition of mupirocin SLN loaded sodium alginate topical gel formulation

Formulation	Zone of inhibition (mm)
F1	22 mm
F2	20 mm
Mupirocin (pure drug)	19 mm
Gentamycin (positive control)	21 mm

CONCLUSION:

The solid lipid nanoparticles loaded gel were successfully developed for topical delivery of mupirocin and tinidazole. AFM imaging of mupirocin-tinidazole loaded SLNs exhibited spherical shape of SLNs. Drug release from SLNs dispersion and SLNs based gel showed sustained release of drug over prolonged period of time as compared to free drug. Drug release from SLN dispersion followed Korsmeyer–Peppas model, indicating fickian diffusion-drug release, while drug release from SLNs based gel followed non fickian model. Stability studies indicated significant change in particle size when stored at room temperature. Developed mupirocin-tinidazole loaded SLNs based formulation may reduce the bacterial burden in a sustained manner. SLN based gel formulation containing sodium alginate was suitable for topical application as it shows much better result for topical antibacterial activity like that of standard similar antibacterial activity like standard drug gentamycin.

ACKNOWLEDGEMENT:

I wish to express my deep sense of gratitude and profound indebtedness to our principal, vice principal, PSG College of Pharmacy and PSG Sons and Charities for providing necessary facilities for doing the project work

CONFLICT OF INTERESTS:

Declared none.

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Received on 18.01.2020 Modified on 29.01.2020

Research J. Science and Tech. 2020; 12(1):57-64.

DOI: 10.5958/2349-2988.2020.00007.8