1. INTRODUCTION:

Necrotizing fasciitis (NF), generally known as flesh-eating disease, is an infection that results in the death of the body's soft tissue. Its a rare bacterial infection of the soft tissue that is part of the connective tissue system that runs throughout the body. It is an unembellished disease of unexpected onset that spreads rapidly. The most commonly infected areas are the limbs and perineum. Only few of such cases arise from chest and abdomen. Classically, the infection enters the body through a disruption in the skin such as a cut or burn.

Risk factors include poor immune function such as diabetes, obesity, cancer, alcoholism, intravenous drug use, and peripheral vascular disease. NF is triggered by a bacterium (monomicrobial NF) or numerous bacteria (polymicrobial NF) infecting the tissue just beneath the skin (subcutaneous tissue). Upon infection, the bacteria or bacterium spreads via the fascia, producing endotoxins (toxins released as the bacteria die and break apart or are lysed) and exotoxins (toxins released by bacteria as waste) that confines blood supply to tissue (tissue ischemia), digestion of cells by enzymes ensuing in a lesion containing of pus and the fluid remains of dead tissue. Because the blood supply to these tissues becomes diminished, neither antibiotics nor the body’s self-mechanisms to fight infection are able to reach them.

Drugs of choice used in treating necrotizing fasciitis are Clindamycin (26.8%), Vancomycin (25.5%), Meropenem (6.1%), Cilastatin (6.1%), Piperacillin (5.6%), Daptomycin (4.8%), Metronidazole (2.6%), Ampicillin (2.2%), Others (16.9%). Clindamycin is an antibiotic recomeneded for the treatment of a numerous bacterial infections. It can be against some cases of Methicillin-resistant Staphylococcus aureus (MRSA). Clindamycin is used basically for the treatment of anaerobic infections caused by vulnerable anaerobic bacteria, which includes dental infections, and infections of the respiratory tract, skin, and soft tissue. Nanosponge is a nascent and developing technology which offers controlled drug delivery for topical use. Nanosponges are minute sponges with a dimension of a virus with an average diameter below 1μm.These tiny sponges has the capability to circulate around the body until they encounter the specific target site and stick on the surface and began to release the drug in a controlled and foreseeable manner^3,4,5. They provide prolonged release as well as improving drugs bioavailability. It is a tiny mesh-like structure in which a large variety of substances can be encapsulated. Nanosponges releases the drug to specific targeted site instead of circulating throughout the body thereby making it more effective for treating necrotizing fasciitis. As Clindamycin is the first choice of drug for the treatment of necrotizing fasciitis and it has low topical bioavailability of 4-5% hence, the bioavailability can be increased by giving nanosponges which will have direct targeting for cell eating bacteria and also give sustained release of the drug over a period of time.

2. MATERIALS:

Clindamycin Phosphate was a gift sample from Mylan laboratory Ltd. Ethyl cellulose (SD Fine Chemicals Ltd., Mumbai), Dichloromethane, Triethanolamine, Di-sodium hydrogen orthophosphate, Glycerine Propylene glycol, Potassium dihydrogen orthophosphate were procured from SD Fine Chemicals Ltd., Carbopol, Methanol from Himedia Laboratory Pvt. Ltd, Mumbai, Sodium hydroxide pellets from Qualigens fine chemicals, Carbopol (Himedia Laboratory Pvt. Ltd, Mumbai), Sodium hydroxide pellets (Qualigens fine chemicals, Mumbai), Di-sodium hydrogen phosphate (SD Fine Chemicals Ltd., Mumbai), Glycerine (SD Fine Chemicals Ltd., Mumbai), Propylene glycol(SD Fine Chemicals Ltd., Mumbai), Dialysis Membrane(Hi - Media Ltd., India).

3. METHODOLOGY:

a. Preformulation Studies

UV-visible Spectrophotometric Method:

A Shimadzu UV-1700 double beam UV-Visible spectrophotometer was used for all measurements. The absorption spectra were recorded over the wavelength range of nm 400 – 200nm, against a solvent blank, in quartz cuvettes with a width of 1cm. The linearity of the calibration curves and the obedience of the method to Beer’s law are authenticated by the high value of the correlation coefficient. The absorption maxima (λ _max) for pure Clindamycin Phosphate were found to be 202 nm which is within the specified limit.

a. Formulation studies-Preparation of Drug Loaded Nanosponges:

Nanosponges were prepared by emulsion solvent diffusion method^1,2,3,4 using different proportion of ethyl cellulose and polyvinyl alcohol. The dispersed phase containing ethyl cellulose and drug was dissolved in 20ml dichloromethane and slowly added to polyvinyl alcohol in 100ml of aqueous continuous phase. The reaction mixture was homogenized using Ultra Turrax followed by sonication using Probe Sonicator for 20mins. The nanosponges formed were then lyophilized and stored in a well closed container. In order to obtain the most satisfactory nanosponge formulation, different formulation parameters such as concentration of retardant material i.e ethyl cellulose was varied from 0.05, 0.2 7 0.15%, surfactant i.e Polyvinyl Alcohol from 0.5, 1.5 to 2.5, volume of external phase from 50, 100 and 150 ml and internal phase from 10, 20 and 30 were standardized keeping all other parameters constant one at a time in trial and error basis. The effect of process parameters such as stirring speed and time of sonication was also studied. The time for homogenization and sonication was kept constant throughout the study i.e, 30 mins and 20 mins respectively. The detailed study of formulation is stated in Table-1.

Optimization using Minitab Software⁵

The optimization of the surfactant concentration and the drug polymer ratio was carried out by Mini tab using Factorial Design.

Table-1 Standardization of formulation parameters

Formulation	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12
Clindamycin Phosphate (gm)	10	10	10	10	10	10	10	101	10	10	1010	10
EC (%)	0.05	0.1	0.15	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Internal phase (ml)	20	20	20	20	20	20	20	20	20	10	20	30
External phase (ml)	100	100	100	100	100	100	50	100	150	100	100	100
PVA (%)	2.5	2.5	2.5	0.5	1.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5

b. Physicochemical characterization of Clindamycin Phosphate loaded nanosponges^6,7,8

Determination of Drug Content:

Nanosponge equivalent to 10mg of Clindamycin Phosphate were dissolved and made up to the mark in 50ml volumetric flask with methanol, further 10ml was diluted to 100ml with methanol and the final dilution were made using Methanol to get a concentration within Beer’s range. The absorbance was measured spectrophotometrically at 202nm using blank nanosponges treated in the same manner as sample. The results obtained were in triplicate.

Drug loading efficiency:

The free drug content was determined by washing the nanosponge with methanol and the filtrate was collected and analysed spectrophotometrically at 202nm.

The loading efficiency (%) was calculated in accordance to the following equation.

Actual Clindamycin phosphate content in nanosponges

Efficiency of = ------------------------------------------------ X 100

Loading Theoretical Clindamycin phosphate content

Percentage yield:

The percentage yield of the nanosponge was calculated accurately by taking the initial weight of the raw materials and the final weight of the nanosponge obtained.

Practical weight of nanosponges

Percentage yield = -------------------------------------------- X 100

Theoretical weight

Particle size:

The mean particle size of nanosponge was measured by Malvern Zeta sizer (Malvern Instrument Ltd). The dispersions were diluted with Millipore-filtered water to an appropriate scattering intensity at 25°C and sample was placed in a disposable sizing cuvette and the particle size were analyzed.

Scanning electron microscopy:

Scanning electron microscopy (SEM) is an electron optical imaging procedure that provides exact photographic images and essential information. SEM is useful for illustrating the morphology and size of microscopic samples with particle size as low as 10-12 grams. The sample was located in an evacuated chamber and scanned in a controlled manner by an electron beam. Interaction of the electron beam with the sample produces a diversity of physical phenomena that, when detected, are used to form images and provide elemental data about the specimens. Scanning electron microscopy (JSM 840A) was used to study the surface morphology of the nanosponges of optimised formulation. The samples were examined after they were gold sputtered by means of 25nm gold film thickness.

In vitro drug release and its kinetics:

Diffusion studies were carried out using Franz diffusion cells with 38ml of receptor cell volume and 5ml donor compartment. The receptor compartments were filled with phosphate buffered pH 7.4 which simulated the physiological pH. Study was carried out using treated cellophane membrane. The entire setup was placed on a thermostatic magnetic stirrer and the temperature was maintained at 37⁰±0.5⁰C throughout the study. Donor compartments were filled with 5ml of 0.4gm of NS equivalent to 0.1gm of Clindamycin Phosphate in phosphate buffer pH 6.8 solutions and covered with aluminium foil to prevent evaporation of vehicle. At regular time intervals; 0.5, 1, 2…24 hours, samples were withdrawn followed by replacement with fresh receptor solution. The drug content in the sample was quantified by UV-Spectrophotometric method. In order to analyze the drug release mechanism, release kinetics was also investigated the outcomes of in vitro drug loaded nanosponges were computed using various kinetic models viz.

Model fitting was accomplished using the PCP DISSO software.

c. Preparation of Nanosponges based hydrogel⁷:

To obtain a suitable topical formulation for application, nanosponges (F22) were incorporated into a gel base. After the preliminary tests for the selection of suitable polymer for gel, Carbopol was found to be ideal. Carbopol was soaked overnight with minimum quantity of water and was allowed to swell up. The Carbopol dispersion was stirred using continuous mechanical stirring for 15 mins. The pH of the resulting dispersion was adjusted with Triethanolamine to form a translucent gel. 0.3ml IPM and glycerol-water mixture with the drug loaded nanosponges was added to the gel under stirring to prepare 30g of gel. Clindamycin Phosphate Nanosponges equivalent to 2.5% w/w of drug were dispersed into the gel base. Permeation enhancer was also added. With a view to incorporate Clindamycin Phosphate nanosponges suitable for topical application, different formulation using Carbopol ranging from 0.5 – 1.5% were coded as G1-G3, G4 – G6 were prepared containing different permeation enhancer (propylene Glycol) to investigate the effect on the permeation characteristics of Clindamycin Phosphate Nanosponges. Formulations G1 to G6 were evaluated for their physical appearance and consistency the results are reported in the Table 3.

Homogeneity:

The prepared hydrogels were visually inspected for clarity, colour and transparency. The prepared nanosponges loaded CP gels were also assessed for the presence of any particles. Smears of gels were prepared on glass slide and detected under the microscope for the presence of any particulate matter or grittiness.

pH of the gels:

The pH range for a perfect gel for topical use is 4.5 - 7. If the pH of the gel goes beyond 7.2, it reaches to alkaline state and the gels irritate the skin. One gram of each blank formulation was dispersed in 30ml of distilled water and the pH was measured using a Micropro Gradmate digital pH meter. Average of three determinations was recorded.

Spreadability:

Spreadability experiment was performed with the help of glass slides and a wooden block, which was provided by a pulley at one end. By this method, Spreadability was estimated on the basis of ‘Slip’ and ‘Drag’ characteristics of gels. A bottom glass slide was fixed on this block. An excess of gel (about1gm) of different formulations were placed on the ground slide. The gel was then squeezed in between this slide and another glass slide having the dimension of fixed ground slide. Surplus of the gel was removed off from the edges. The top plate was then subjected to pull of 20gms, lesser the time taken for parting of two slides better was the Spreadability.

Spreadability was then calculated using the following formula:

S = M × L/ T

Where,

S = Is the Spreadability,

M = Is the weight in the pan (tied to the upper slide),

L = Is the length moved by the glass slide

T = Represents the time taken to separate the slide completely from each other.

Extrudability Study:

A locked collapsible tube comprising above 20grams of the gel was hard-pressed at the crimped end and a clamp was applied to avert any rollback. The cap was removed and the gel was extruded till the pressure was dissipated.

Viscosity:

The viscosity of gels is dependent on type and concentration of polymer used. The ideal viscosity of gels varies from 2000 – 6000cps. It is an important parameter in evaluation of physical parameters of gel. As the viscosity reduces, Spreadability and Extrudability also reduces. It also affects the stability of gels. The viscosity of different mucoadhesive gel formulations was determined using a Model DV- III + Programmable Rheometer Brook field viscometer using spindle #7 at 100 rpm with torque ranging from 10-100%. Average of three determinations was recorded.

Percentage yield of the gel:

Weight of all the ingredients used was added up theoretically. Total prepared gel was weighed which was the practical yield. The percentage yield was calculated according to the formulae

% Yield= Practical yield/Theoretical yield *100

Drug Content:

1gm of Clindamycin Phosphate gel was accurately weighed dissolved using methanol, sonicated for a period of 10-15mins and made up to the mark in 10ml volumetric flask with methanol. From this 1ml was pipette out and diluted to 10ml with methanol. From this 0.5ml was pipette and finally diluted to 10ml to get a concentration within Beer’s range. The absorbance was measured spectrophotometrically at 202nm against blank gel treated in the same manner as sample. Average of three readings was recorded.

In-vitro release studies:

Diffusion studies were carried out using Franz diffusion cells with 38ml of receptor cell volume and 5ml donor compartment. The receptor compartments were filled with phosphate buffered pH 7.4 which simulated the physiological pH. Study was carried out using treated cellophane membrane. The entire setup was placed on a thermostatic magnetic stirrer and the temperature was maintained at 37⁰±0.5⁰C throughout the study. Donor compartments were filled with 5ml of 0.4gm of gel equivalent to 01.gm of Clindamycin Phosphate in phosphate buffer pH 6.8 solutions and covered with aluminium foil to prevent evaporation of vehicle. At regular time intervals; 0.5, 1, 2…24 hours, samples were withdrawn followed by replacement with fresh receptor solution. The drug content in the sample was quantified by UV-Spectrophotometric method as mentioned above.

Microbiological Studies:

The standardized final formulation of nanosponge of Clindamycin Phosphate by emulsion solvent diffusion method i.e, F22 and also the standardized gel formulation i.e, G5 and G6 were selected for the microbial studies. The primary objective was to compare anti-bacterial activity of the developed formulations with that of the saline water solution. Muller Hington agar was the nutrient medium used for the study. After hardening the medium by solidification at room temperature, with the help of a sterile cork borer, cups of each 6 mm diameter were pierced and scooped out from the petridish. Using sterile pipettes sample solutions (0.5gm, 1gm) of different formulation equivalent to 10 mcg of the drug were fed into the cup. The petridish was then incubated for 48 hours at 37ºC. After incubation the zone of inhibition was measured.

4. RESULT AND DISCUSSION:

Preformulation Studies:

UV-visible Spectrophotometric Method:

λmax of Clindamycin Phosphate was determined in pH 6.75 and pH 7.4 in UV spectrophotometer (Shimadzu UV-1700 double beam spectrophotometer) by scanning in a wavelength range of 400-200nm. λmax of Clindamycin Phosphate was found to be 202nm. A simple reproducible method of estimation was standardized against the blank. The standard graph obtained was linear, with regression coefficient of 0.998 and 0.997.

Figure 1: UV Spectrum of Clindamycin Phosphate of Clindamycin Phosphate in phosphate buffer pH 6.75 and 7.4 at λ_max was found to be 202 nm.

Formulation of nanosponges by emulsion solvent diffusion method:

In emulsion solvent diffusion method, the formation of the nanosponge is by the rapid diffusion of dichloromethane into the aqueous medium. The instant mixing of the dichloromethane and water (aqueous medium to the polymer medium) induced precipitation of the polymer at the interface of the droplets, thus forming a shell enclosing the dichloromethane and the dissolved drug. The finely dispersed droplets of the drug - polymer solution was solidified in the aqueous phase via diffusion of the solvent. Effect of different formulation parameters were tested via trial-and-error method to get optimized formulation. The polymer used in the formulation of emulsion solvent diffusion method was Ethyl cellulose which acted as a retardant material in the internal phase, Poly vinyl alcohol was the surfactant, Dichloromethane solvent and water as a vehicle the formed.

Optimization by 3² full factorial design:

In order to optimize Drug: polymer ratio and concentration of surfactant, factorial design was adopted. To learn all the probable combinations of both factors at all levels, a two factor, three level full factorial designs was constructed and conducted in a fully randomized manner.

Minitab software was used to apply full factorial design to study response surface of 3 level factorial design with 13 runs in a quadratic model. The formulations were made-up according to a 3² full factorial design, letting the concurrent evaluation of two formulation variables and their interaction. Based on the preliminary studies, the two independent variables included were surfactant concentration and drug: polymer ratio. The two variables were compared over 3 levels, +1(High), 0(Medium) and -1(Low). The effect of the two factors on response of dependant variable i.e, Y₁(Particle Size), Y₂ (Entrapment Efficiency) and Y₃ (P.D.I) was studied by polynomial equation.

Table-3 Physicochemical properties of prepared nanosponges

Sl. No	Formulation Code	Particle Size	P.D.I	% Yield	Drug Content	Free Drug	Entraptment Efficiency
1	F16	384.80	0.129	88.90±0.55	84.89±1.28	8.9	78.98
2	F17	496.20	0.487	88.43±1.11	72.46±1.56	14.9	65.11
3	F18	532.80	0.689	85.81±0.67	72.39±2.64	13.09	54.23
4	F19	168.30	0.586	91.82±1.77	85.48±1.99	9.65	81.34
5	F20	232.40	0.346	89.11±1.90	79.38±2.18	17.9	72.23
6	F21	286.80	0.984	87.38±0.50	75.46±1.67	18.0	69.35
7	F22	72.04	0.137	98.76±2.11	88.76±0.58	7.09	84.13
8	F23	108.40	0.786	96.31±0.32	83.29±1.28	9.19	79.26
9	F24	135.50	0.982	94.30±0.55	79.86±2.42	18.9	73.17
10	F25	90.18	0.123	96.89±2.16	85.17±1.04	8.78	83.14
11	F26	115.6	0.324	95.99±0.77	79.02±0.04	8.99	74.37
12	F27	167.9	0.276	91.76±1.34	77.45±1.78	9.73	78.23
13	F28	105.34	0.195	96.54±1.11	82.23±0.04	8.02	80.11

Determination of Drug Content:

The different batches of the drug loaded nanosponges were exposed for drug content analysis. The powdered nanosponges (10mg equivalent) were dissolved in adequate quantity (100ml) of phosphate buffer PH 6.8 then filter. The UV absorbance of the filtrate was measured using a UV spectrophotometer at 202nm. The drug content of different formulation was found to be in the range of 72.46±1.56 to 88.76±0.58 as shown in below table 8.

Drug loading efficiency:

The loading efficiency (%entrapment) of Clindamycin Phosphate nanosponge formulations are given in table 8. The loading efficiency calculated for all nanosponges ranged from 54.23 to 84.13%. The highest loading efficiency was found for the F22 formulation to be 84%, where a greater amount of drug was encapsulated. The highest loading efficiency, greater the amount of drug was entrapment.

Percentage yield:

The percentage yield of Clindamycin Phosphate nanosponge formulation is given in Table 8. Production yield calculated for all nanosponges ranged from 88.43% – 98.76%. The production yield was found the highest for formulation F22 i.e, 98.76% respectively. From the production yields of Clindamycin Phosphate nanosponge formulation, it was indicated that increasing the drug: polymer ratio increased the production yield.

Particle size and PDI:

Particle size analyses of all the formulation was carried out using Malvern particle size analyzer. All the formulated NSs were in nano-size range with narrow particle size distribution as per PDI. Out of all the factorial formulations (F16 - F28), F22 showed the least particle size of 72.04 nm (Figure 4) , with P.D.I of 0.137 and showed highest entrapment efficiency. The average particle size of nanosponges can be highly influenced by drug: polymer ratio. The low concentration of polymer selected improves the diffusion of dichloromethane (internal phase) into aqueous phase (external phase) hence giving less time for the droplet formation and so it decreases the particle size. The F22 formulation is the same as F1 formulation which was done in the Preformulation trials.

Figure 2: Particle Size and PDI of F22 Fomulation

Scanning electron microscopy:

The morphology of the optimized nanosponge prepared by emulsion solvent diffusion method F22 was investigated by SEM. The representative SEM photographs of the nanosponges are shown in figure 5. represents the spherical shape of NanoSponges with nanosize range. It was projected that the in-ward diffusion of DCM on the EC polymeric surface contributed to the porus spongy nature of the nanosponges. Also, the micrographs (Figure 5) revealed that the EC matrix was properly coated over the cp (clindamycin phosphate) and spherical also glazed by PVA responsible for anti-adhesiveness between the particles and smooth surface of Nanosponges.

Figure 3: SEM of Optimized Formulation

In-vitro Drug release:

Diffusion studies were carried out using Franz diffusion cells with 38 ml of receptor cell volume and 5 ml donor compartment. The receptor compartments were filled with phosphate buffered pH 7.4 which simulated the physiological pH. The % release of the two optimized formulations (F22 and F25) was studied for 24hours (Table 9). It was found that formulation containing polymer and retardant material in 1:0.5 ratio (F28) has shown maximum in vitro drug release as compared to other formulations. The drug release was found to be highest for F22 i.e, 83.5%. this could be attributed to the highest degree of the encapsulation of drugs in the inner structure of the NS. Figure 6 shows the Comparison Drug release studies of F22, F23. Additionally, release profiles of all the formulations have been subjected to diverse release kinetics mathematical models; The interpretation of data of the drug showed that maximum linearity (highest R2 value) was found with Higuchi classical model of diffusion; (R2 = 0.977) as shown in Table 4. This manifests that the drug release mechanism is by diffusion process. To determine whether this diffusion process is Fickian or non Fickian, the Korsmeyer-Peppas model was applied to 60% of the release data. The results obtained after the application of Korsmeyer-Peppas model showed exponent (0.45 < n < 0.89) ‘n’ values to be less than 0.5 which suggests that the diffusion process is obeying Fick’s law of diffusion, which revealed that Korsmeyer Peppas model is the best fit model for F22 with a regression coefficient (r²) value of 0.997. Moreover, the Korsmeyer–Peppas model suggested all nanosponges represented non swellable matrix diffusion drug release mechanism attributed to porosity in these nanosponges formulations. Based on the above results, F22 formulation was found to have maximum EE and in vitro drug release. Henceforth further characterization studies were performed on this formulation.

Table 4: % Drug Release studies of the nanosponges

SL. No	Time	%CDR
SL. No	Time	F22	F25
1	0	0	0
2	0.5	5.87 ± 1.28	4.36 ± 0.12
3	1	10.76 ± 1.21	11.32 ± 0.21
4	2	20.68 ± 0.19	23.05 ± 0.14
5	3	33.41 ± 0.12	30.56 ± 0.16
6	4	39.86 ± 0.09	37.12 ± 0.16
7	5	43.34 ± 0.11	45.13 ± 0.26
8	6	50.86 ± 0.34	54.83 ± 0.24
9	7	65.89 ± 0.12	63.92 ± 0.20
10	24	83.5 ± 0.12	77.7 ± 0.21

Gel loaded Nanosponge:

Based on the particle size, % entrapment and % release F22 nanosponge formulation was selected for further incorporation into a topical gel. Gels were formulated using Carbopol 934 as the gelling agent. It was found that 1% Carbopol 934 was ideal for the formation of effective and satisfactory gel. Lower concentrations of (0.5%) or higher concentration (1.5%) of Carbopol resulted in a non-consistent gel. As our objective was to achieve greater permeability hence formulation G5 and G6 was finalized which had Permeation enhancers incorporated in them. The formed Hydrogels were evaluated for Physicochemcial properties.

Homogeneity:

The prepared gel formulations G5 and G6 were found to be translucent and homogenous. It didn’t give any gritty feeling on applying on the skin surface

pH:

In gel formulations pH is maintained to simulate the neutral condition of the skin. The pH of the prepared gel formulations G5 and G6 was found to be in the range of pH 6.52 -6.79.

Viscosity:

Based on the viscosity of the polymer, the ideal viscosity of gels varies from 2000 – 6000cps. The viscosity of prepared gel formulations G5 and G6 was evaluated using Brooke filed viscometer and their viscosity varied from 4500cps - 5356cps.

Spreadability:

The therapeutic efficiency of the formation also depends on its spreadability values. The prepared gel formulations G5 and G6 were evaluated for Spreadability and the results showed that Spreadability varied from 8.28gm.cm/sec to 10.56gm.cm/sec respectively.

Extrudability:

The prepared gel formulations G5 and G6 were evaluated for Extrudability. G6 showed good Extrudability behaviour.

Drug content:

The drug content of G5 was found to be 85.79% and G6 to be 91.8 % respectively.

Table 6: Evaluation of standardized nanosponge gels

Sl. No	1	2
Formulation code	G5	G6
Appearance and homogeneity	Gel like consistency, very good homogeneity, Translucent	Gel like consistency, very good homogeneity, Translucent
pH ± SD	6.52	6.79
Viscosity ± SD (cps)	4500 ± 0.2449	5356 ± 0.1476
Spreadability (gm.cm/sec)	8.28	10.56
Extrudability	Good flow	Excellent flow
% Yield ± SD	92.63	93.63
Drug content (%) ± SD	85.79 ± 0.0147	91.8 ± 0.0121

In-vitro release studies of gels:

The % releases of the standardized nanosponge gel G5 and G6 were studied for 24 hours. The gels showed sustained release for the period of 24hours. % release for the 1^st hour was 9.39% and 10.48% and for the 7^th hour was 64.86% and 70.89% and for 24^th hour was 84.45% and 88.96% (Table 12).

Table7: In Vitro Release Study of the Formulated nanosponge loaded Gels

Sl. No	Time	%CDR
Sl. No	Time	G5	G6
1	0	0	0
2	1	9.39 ± 0.27	10.48 ± 0.20
3	2	12.31 ± 0.098	14.38 ± 0.3710
4	3	22.36 ± 0.231	23.92 ± 0.102
5	4	36.1 ± 0.231	35.89 ± 0.278
6	5	45.47 ± 0.156	50.38 ± 0.2298
7	6	53.13 ± 0.121	66.1 ± 0.288
8	7	64.86 ± 0.202	70.89 ± 0.222
9	24	82.45 ± 0.512	88.96 ± 0.212

Microbiological Studies:

Microbiological studies were carried out for the saline solution with the optimized gels loaded with nanosponges. Muller Hington agar was the nutrient medium and staphylococcus aureus was the strain organism used for the study. After incubation for about 24hours, zone of inhibition were measured for the standard and gels. The zones of inhibition was seen the highest i.e, 40.4mm for the gel formulation (G6). The formulated nanosponge gels G6 showed better antibacterial activity.

Table 8: Zone of inhibitions of nanosponges loaded gels of G5 and G6

Sl. No	Formulation Code	Inoculums	Weight of the gel (gm)	Zone of inhibition (nm)
1	G5	0.05	0.5	38.6
2	G6	0.05	1	40.4
3	G5	0.1	0.5	33.9
4	G6	0.1	1	35.1

Figure 4: Depicting the zone of inhibition for sample Clindamycin Phosphate which contains 0.5 gm , 1 gm of gel which has 0.05 ml and 0.1ml of inoculums.

5. CONCLUSION:

The present study was an effort to design, develop, and evaluate the Clindamycin Phosphate loaded nanosponges incorporated in gel to achieve topical drug delivery of the drug for sustained release and increased bioavailability. In addition it was decided to formulate a drug delivery system which would achieve a prolonged release of the drug. Nanosponges are miniature sponges with a size of a virus that circulate around the body till they meet the specific target site, then stick onto the surface and start to release the drug in a controlled and foreseeable manner. The best standardized formulation obtained by the emulsion solvent diffusion method i.e, F22 showed good loading efficiency of 84.13%, production yield of 98.76%, particle size of 72.04 nm and drug content was 85.17%. F22 was incorporated into standardized Carbopol based gels (G5 and G6). Evaluation of the formulated gels G5 and G6 showed a production yield of 93.53% and 94.63% and drug content of 85.79% and 91.8% respectively. The nanosponge based formulation G6 showed better drug release and good have better penetration along with better anti-bacterial effect hence we can speculate that Clindamycin Phosphate nanosponge loaded hydrogel formulation is a good candidate for topical drug delivery in the treatment of necrotizing fasciitis.

6. AUTHOR CONTRIBUTION:

All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission

7. CONFLICT OF INTEREST:

The authors declare that no conflict of interest is associated with this work.

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Received on 29.07.2022 Modified on 15.11.2022

Research J. Pharm. and Tech 2023; 16(10):4626-4634.

DOI: 10.52711/0974-360X.2023.00753

Formulation Code	Carbopol 934 Concentration (%)	Triethanolamine (w/v)	Clindamycin Phosphate Nanosponges (w/w)	Propylene Glycol
G1	0.5	0.5%	1%	--------
G2	1.0	0.5%	1%	--------
G3	1.5	0.5%	1%	---------
G4	1	0.5	1%	5
G5	1	0.5	1%	10
G6	1	0.5	1%	15