formulation and evaluation of tetracycline …

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Sorlin et al. World Journal of Pharmacy and Pharmaceutical Sciences

FORMULATION AND EVALUATION OF TETRACYCLINE

NIOSOMAL TOPICAL GEL DRUG DELIVERY SYSTEM

Sorlin Selva Joice P.*1, Rubina Reichal C.

2, Thirumoorthy N.

2 and Sangeetha M.

2

1Assistant Professor, Department of Pharmaceutics, Cherraan’s College of Pharmacy,

Coimbatore, Tamil Nadu, India.

2Department of Pharmaceutics, Cherraan’s College of Pharmacy, Coimbatore, Tamil Nadu,

India.

ABSTRACT

The core objective of the present study is to formulate Tetracycline

niosomes for topical delivery system by hand shaking method. The

prepared niosomes are to be characterized for their size, shape,

stability, entrapment efficiency, invitro drug release and retention

study. The formulation 1(F1) was more stable when compared to other

formulations with smaller size vesicles and showed higher entrapment

efficiency. The best formulation was prepared as gel with suitable gel

base. The Tetracycline Niosomal gel delivery may reduce the

frequency of dosing intervals and improve patient compliance.

KEY WORDS: Tetracycline, Niosomal Gel, Topical Drug Delivery,

Acne Vulgaris.

INTRODUCTION

Even the oral route is the convenient route for delivery of drugs, it has some limitation in the

treatment of skin diseases. The topical delivery system has better percutaneous absorption

than the semi solid preparations.[1-4]

Recently niosomes are becoming popular in the field of

topical drug delivery due to its outstanding characteristics like enhancing the penetration of

drugs, providing a sustained pattern of drug release and ability to carry both hydrophilic and

lipophilic drugs. Topically applied Niosomes can increase residence time of drug in the

stratum corneum and epidermis. Topical applicability of niosomes was further enhanced by

developing niosomal gel formulation using carbopals.[5,6]

The release from the niosomal gel

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 8, 2644-2657 Research Article ISSN 2278 – 4357

Article Received on

20 June 2017,

Revised on 11 July 2017, Accepted on 1 August 2017,

DOI: 10.20959/wjpps20178-9963

*Corresponding Author

Sorlin Selva Joice P.

Assistant Professor,

Department of

Pharmaceutics, Cherraan’s

College of Pharmacy,

Coimbatore, Tamil Nadu,

India.


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was highly prolonged when compare to conventional gel formulation. As well as the presence

of other ingredients that act as skin permeation co-enhancers.[7,8]

Chemically Tetracycline is (4S,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a-

pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

(Fig No.1 ) inhibits protein synthesis by binding to 30S ribosomes the attachment of

aminoacyl- t-RNA to the acceptor A site of mRNA ribosome. As a result, the peptide chain

fails to grow10,11

. The action is usually inhibitory and reversible upon withdrawal of the drug.

Mammalian cells are less vulnerable to the effect of tetracyclines, despite the fact that

tetracycline binds to the small ribosomal subunit of both prokaryotes and eukaryotes (30S

and 40S respectively). This is because bacteria actively pump tetracycline into their

cytoplasm, even against a concentration gradient, whereas mammalian cells do not. This

accounts for the relatively small off-site effect of tetracycline on human cells.

Fig. 1: Chemical Structure of Tetracycline.

It is a broad spectrum antibiotic, used to treat Acne Vulgaris, Syphilis, Chlamydia infection,

and Bronchitis.

In the novel drug delivery system, there is number of permeation enhancers has been

introduced to effective plasma drug level, in this niosomes play a vital role in topical drug

delivery. The presence of nonionic surfactants increases the permeability of Tetracycline

through biological membrane and also reduces the systemic toxicity. The niosomal gel of

tetracycline may increase efficacy for sustained activity.

MATERIALS AND METHODS

Tetracycline was obtained as a gift sample from Emil Pharma, Maharastra.All other

chemicals used were of analytical grade.


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Preformulation Studies

Compatibility Studies

Drug and excipients interaction study is carried out by FTIR technique.

Preparation of Niosomes

Cholesterol and span 20 were taken in specified ratios of (1:1, 1:1.5,1:2,1:2.5,1:3 & 1:3.5)

and transferred in to a clean round bottom flask. Then the lipid mixture was dissolved in 10

ml of diethyl ether. The flask was continuously vortexed to form a thin film along the sides of

the flask. An appropriate amount of tetracycline was dissolved in phosphate buffer saline

(PBS) pH.7.4. This was poured into the thin film and vortexed continuously for a period of

30 min at room temperature.[4,6]

Table 1: Composition of the Tetracycline Niosomal formulations.

Evaluation of Prepared Tetracycline Niosomes

By optical microscopy

Take one drop of prepared niosomes (diluted with water if needed) in a clean glass slide; it is

then placed on the stage of the optical microscope. First it was viewed under low power then

changed to high power and the vesicles were counted up to 100 numbers.

By scanning Electron microscopy (SEM)

The size of the vesicles was measured by scanning electron microscopy (HITACHI S – 150).

Percentage Encapsulation of drug

Tetracycline encapsulated niosomes were separated from unentrapped drug by dialysis

method for 24 hrs. The formulation were transferred into a standard flask, then lysed with1ml

of 2.5% SLS solution, then incubated at 370 1

0 C for 2 hrs. Then it is filtered through

whatman filter paper. Filtrate of 1 ml was diluted to 10 ml with PBS and absorbance of the

resulting solution was measured spectrophotometrically at 255 nm.

Formulation Code Drug Span 20 Cholesterol Diethyl ether

F1 100 mg 10 ml 10 mg 10 ml

F2 100 mg 15 ml 10 mg 10 ml

F3 100 mg 20 ml 10 mg 10 ml

F4 100mg 25ml 10mg 10ml

F5 100mg 30ml 10mg 10ml

F6 100mg 35ml 10mg 10ml


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Determination of percentage of drug encapsulated in the niosomes

This was carried out to find out the percentage of drugs encapsulated in the niosomes by

using the following formula:

Drug leakage studies from vesicles

Vesicle stability with respect to drug leakage and drug degradation upon storage was studied

at refrigeration (40c), room temperature (25

0c) and high temperature (37

0c) for a period of

one month on niosomal suspension and gel samples containing a known amount of

Tetracycline, contained in light resistant containers. Samples were withdrawn at weekly

intervals, and entrapment efficiency was determined.

In Vitro Release Pattern of Tetracycline Niosomal Formulations

The niosomal gel preparation was taken in a dialysis tube, which acts as a donor

compartment. Dialysis tube was placed in a beaker containing 250 ml of phosphate buffer

saline of pH 7.4, which acts as a receptor compartment diffusion medium was replaced after

every withdrawal so that the volume of the diffusion medium was maintained. The collected

samples were analysed at 255nm.[11,12]

Formulation of Gel

Carbopol 940 was soaked in water for overnight, then required quantity of niosomal

suspension was dispersed in solution with stirring and required amount of tri ethanolamine

and glycerine was slowly added with stirring to obtain a clear gel.[16,17]

Table 2: Composition of the Tetracycline Niosomal gel Formulation.

Ingredients For 25 gm

Tetracycline niosomal suspension 0.25 ml

Carbopol 0.25 gm

Tri ethanolamine 0.125 ml

Glycerol 2.5 ml

Purified water 21.8 ml

Evaluation of Tetracycline Niosomal gel formulation

The formulated gel was examined for following tests.


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Physical appearance

The prepared gel was examined for clarity, color, homogenicity and the presence of

aggregates.

pH

2.5 g of gel were accurately weighed and dispersed in 25 ml of distilled water. The pH of the

dispersion was measured by using a digital pH meter.

Rheological study

Viscosity was determined by Brookfield viscometer.

Drug content

Tetracycline encapsulated niosomal gel were transferred into a standard flask, then lysed

with1ml of 2.5% SLS solution, then incubated at 370 1

0 C for 2 hrs. Then it is filtered by

using whatman filter paper. Filtrate of 1 ml was diluted to 10 ml with PBS and absorbance of

the resulting solution was measured spectrophotometrically at 255 nm.

In vitro drug diffusion study

The niosomal gel preparation was taken in a dialysis tube, which acts as a donor

compartment. Dialysis tube was placed in a beaker containing 250 ml of phosphate buffer

saline of pH 7.4, which acts as a receptor compartment diffusion medium was replaced after

every withdrawal so that the volume of the diffusion medium was maintained. The collected

samples were analysed at 255nm.[13]

Study of Release Kinetics

The optimized formulation F1 was subjected to graphical treatment of assesses the kinetics of

drug release. The data obtained from the best formulation were fitted to various kinetics

equations (first order, second order, higuchi’s and koresmeyer’s –peppas model) to determine

the mechanism of drug release and release rate as indicated by higher correlation co efficient

(r2).

Stability Studies




0c) for a period of



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RESULTS AND DISCUSSION

FT-IR Studies

The IR Spectrum of pure Tetracycline drug was compared with the IR spectrum of physical

mixture of drug and excipients.

Fig. 2: FTIR Spectrum of Tetracycline.

Fig. 3: FTIR Spectrum of Carbopol.

Fig. 4: FTIR Spectrum of Cholesterol.


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Fig. 5: FTIR Spectrum of Tetracycline Carbapol.

Fig. 6: FTIR spectrrum of Tetracycline Cholesterol.

From the spectra, (Fig 2-6) there is no appearance or disappearance of any characteristics

peaks. This shows that there is no interaction between the drug and excipients used in the

vesicles preparation.

Table 3: Size Distribution of Niosomes by Hand Shaking Method.

Size range

(m)

Number of Niosomes

F 1 F 2 F 3 F 4 F 5 F 6

Below 0.1 18-20 16-18 8-9 18-20 13-15 6-8

0.1-5 70-72 72-75 85-87 65-70 68-74 82- 86

Above 5 13-17 6-9 5-9 3-6 4-6 4-5

Optical Microscopic view of Tetracycline loaded Niosomes prepared by Hand Shaking

Method

The optical microscope of the prepared niosomes reveals that they are discrete and spherical

in shape. The vesicles are slightly black in color. There is a thickening around the inner


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compartment of the vesicles prepared by hand shaking method. Individual vesicles could be

seen under high power as shown in Fig. 7.

Fig. 7: Optical Microscopic view of Tetracycline Loaded Niosome.

By scanning electron microscopy (SEM)

The prepared niosomes vesicles sizes were performed by SEM. The size range was observed

to be 0.5-5m. This is shown in Fig.8.

Fig. 8: Scanning Electron Microscopic view of Tetracycline Loaded Niosomes.

Fig. 9: Scanning Electron Microscopic view of Tetracycline Loaded Niosomal Gel.


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Drug Retention Studies

Table 4: Percentage of Drug Retention in Tetracycline Niosomal formulation.

Formulation

Code

Percentage of drug retention in niosomes

Refrigeration

Temp. (40 1 C)

Room Temp

(250 1 C)

High Temp.

(370 1 C)

Days Days Days

7 14 21 28 7 14 21 28 7 14 21 28

F1 100 95 90 86 99 88 84 77 94 85 73 69

F2 100 96 95 87 99 90 84 79 95 84 75 70

F3 100 97 95 90 100 94 87 80 95 85 76 70

F4 100 95 93 85 98 93 84 77 93 84 73 68

F5 100 97 94 86 99 95 85 78 94 85 74 69

F6 100 98 95 90 100 95 88 80 96 86 75 72

From the results, Niosomal formulation F1 showed good retention when comparing with

other formulations.

Table 5: Percentage of Drug Retention in Tetracycline Niosomal Gel formulation.

Formulation

Code

Percentage of drug retention in Niosomal Gel

Refrigeration

Temp. (40 1 C)

Room Temp

(250 1 C)

High Temp.

(370 1 C)

Days Days Days

7 14 21 28 7 14 21 28 7 14 21 28

Tetracycline

Niosomal Gel 100 99 98 96 98 90 86 81 96 86 79 70

The results of the prepared niosomal gel (F1) had showed good retention.

Fig. 10: Drug Retention Plot for Tetracycline Niosomal Gel.

Percentage of Drug Encapsulation

The percentages of drug encapsulated in niosomes are given in the Table 6. The Percentage

of drug encapsulation varies when the Span 20 proportion was varied. This explained that

encapsulation was increased with decrease in Span 20 content while cholesterol content was

maintained at constant value.


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Table 6: Encapsulation Efficiency for Tetracycline Niosomal Formulations.

Formulation Code Amount of drug used in (mgs) Percentage of drug encapsulated

F 1 100 mg 88.63

F 2 100 mg 81.34

F 3 100 mg 71.56

F 4 100 mg 70.23

F 5 100 mg 79.67

F 6 100 mg 86.29

Invitro Drug Release Study

In vitro drug release was studied for all the batches of niosomes. The studies were performed

up to 12 h for all the batches. The results show that the percentage of drug release at 12hrs of

F1 was found to be 61.40. It confirmed that F1 showed the sustained action.

Table 7: In vitro Dissolution Study of Trial Formulations of Tetracycline Niosomes.

Time F1 F2 F3 F4 F5 F6

0 0 0 0 0 0 0

2 16.48 18.55 19.62 20.3 17.69 17.29

4 26.34 28.37 32.64 36.94 29.8 30.47

6 29.56 43.78 47.56 48.78 34.38 53.49

8 42.24 55.04 65.76 64.87 47.8 72.88

10 56.74 68.8 72.78 73.28 62.48 84.93

12 61.40 74.89 84.23 87.18 70.08 93.73

Fig. 11: Invitro % Drug release of Trial formulations of Tetracycline Niosomes.

Study of Release Kinetics

Studies revealed that the release of drug from niosomes F1 formulation were found to be

Zero order kinetic indicating that the concentration was independent of drug release .More

over Korameyer – Peppas model indicated a good linearity(r2=0.989) and Peppas model was

0.5< n which implies that the drug follows Non-Fickian transport. From this study it was

found that the niosomal F1 formulation was diffusion controlled.


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Fig. 12: Zero Order Release . Fig. 13: First Order Release.

Fig. 14: Higuchi Model. Fig. 15: Korsmeyer’s Model.

Stability Studies




0c) for a period of




Table 8: Stability Studies.

Temperature

Amount of Drug Retained (%) ± S.D

Initial After 1

month

After 2

months

After 3

months

After 4

months

After 5

months

After 6

months

Refrigeration

(4°±1°C) 99.68±0.44 98.24±0.16 96.2±0.36 95.74±0.41 94.83±0.67 94.06±0.28 94.02±0.62

Room

Temperature

(25°C±2°C,

60%±5% RH)

99.68±0.44 97.17±0.34 95.36±0.23 93.74±0.72 93.24±0.36 93.18±0.81 92.17±0.73


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Fig. 16: Amount of Drug Retained.

Table 9: Invitro Release Profile of Optimized Formulation Before and After Storage at

Room Temperature (25°±2°C, 60%±5% Rh) for 6 Months.

Time in Hours Cumulative Percentage of Drug Released ±S.D

Before Storage After Storage

0.25 4.88±0.28 4.25±0.24

0.50 6.01±0.31 6.08±0.81

0.75 11.06±0.51 9.45±0.13

1.00 15.03±0.42 14.24±0.31

1.50 19.41±0.72 17.36±0.42

2.00 25.95±0.83 24.23±0.62

2.50 29.2±0.62 28.24±0.16

3.00 32.5±0.54 33.90±0.12

4.00 37.24±0.43 36.51±0.71

5.00 41.09±0.47 39.03±0.59

6.00 46.37±0.32 45.64±0.68

7.00 51.02±0.67 50.82±0.37

8.00 55.95±0.14 54.42±0.58

9.00 61.05±0.26 59.08±0.53

10.00 66.15±0.19 65.47±0.41

11.00 71.61±0.54 69.22±0.52

12.00 74.64±0.32 70.50±0.81

13.00 76.51±0.41 72.74±0.39

14.00 76.98±0.58 73.23±0.73

15.00 78.00±0.61 76.78±0.37

16.00 78.04±0.32 76.19±0.86

17.00 77.25±0.59 76.05±0.31

From these results, it was observed that there was no change in the drug release before and

after storage.

CONCLUSION

Niosomal gel drug delivery has been applied for topical application to increase the skin

penetration and skin retention. The prepared tetracycline topical gel can used for the


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treatment of Acne Vulgaris and showed good entrapment efficacy with smaller vesicle size.

From this it was concluded that the prepared tetracycline niosomal gel drug delivery can

improve the patient compliance and showed sustained activity.

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