Development, Characterization and Evaluation of Niosomes and Liposomes of Bacitracin Zinc.

 

Derle D.V., Kasliwal N.H., Gandhi P.P. and Yeole D.R.

Department of Pharmaceutical Sciences, N.D.M.V.P. Samaj’s, College of Pharmacy, Gangapur Road, Nasik- 422 002, Maharashtra.

*Corresponding Author E-mail: dvderle@yahoo.com

 

ABSTRACT:

The skin permeation of antibacterial agent, bacitracin zinc, in liposomes and niosomes, after topical application, were elucidated in the present study with aimed to increase its penetration capacity hence efficiency. The formulations of bacitracin zinc were prepared and characterized for vesicle size, entrapment efficiency, and drug permeation across rat skin and were evaluated for their stability. Formulation with niosomes demonstrated a better skin permeation potential, sustained release characteristics and higher stability as compared to liposomes. The ability of liposomes and niosomes to modulate drug delivery makes the two vesicles useful to formulate topical bacitracin zinc.

 

KEYWORDS: Bacitracin zinc, Liposomes, Niosomes, Skin permeation

 


INTRODUCTION:

Liposomes are the lipid vesicles prepared from a variety of natural and synthetic phospholipids are being considered as drug carrying structures. They may serve as a solubilization matrix, as local depot for sustained release of dermally active compounds, as permeation enhancers, or as a rate-limiting membrane barrier for the modulation of systemic absorption of drugs via the skin (Schreier and Bouwstra, 1994). Thus, it has been widely investigated in topical applications for skin (Valenta et al., 2000; Touitou et al., 1994; Kuroski, et al., 1991). Niosomes, non-ionic surfactant based vesicles are formed from the self-assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer structures which can entrap both hydrophilic and lipophilic drugs either in an aqueous layer or in vesicular membrane (Uchegbu and Vyas, 1998; Carafa et al., 2002). These structures are analogous to liposomes. The low cost, greater stability, ease of storage and also large number of available vesicle forming non-ionic surfactants has lead to exploitation of these compounds as alternatives to phospholipids for industrial production both in pharmaceutical and cosmetic applications (Vora et al., 1998; Varshosaz et al., 2003).

 

 

Preliminary studies indicate that niosomes behave in vivo like liposomes, prolonging the circulation of entrapped drug to alter its organ distribution and metabolic stability, or prolonging the contact time of drug with the applied tissues in topical application (Manconi et al., 2001; Shahiwala and Misra, 2002; Vora et al., 1998), which demonstrated that niosomes could improve drug skin penetration and increase its accumulation in the superficial skin strata.

 

Bacitracin zinc is an antibacterial agent, which is mainly used in the treatment of ophthalmic and dermatological infections. In most cases of primary and secondary skin bacterial infections, the disease treatment by topical drug application is not sufficient, and systemic antibiotic treatment is required. The systemic antibiotic treatment is associated with severe allergic reactions, headache, and serious nephrotoxicity. Furthermore, drug gets inactivated on passage through the gastrointestinal (GI) tract due to degradation (Mcleney and Johnson, 1949; Greenberg et al., 2007). Transdermal delivery of bacitracin zinc is a better option to overcome problems associated with its parenteral delivery. Commercial conventional such as creams, ointments are available; however, absorption of the bacitracin zinc is limited by poor penetration through the stratum corneum and is also limited by its very low water solubility, requiring it to be incorporated into a suitable vehicle.  The entrapment of drug in vesicles may help in the localized delivery of the drug, and an improved solubility and availability of the drug at the site may reduce systemic side effect. However, there is no investigation reported on bacitracin zinc loaded niosomes in topical drug delivery.

 

The purpose of the current study was to investigate the feasibility of liposomes and niosomes to formulate the transdermal administration of bacitracin zinc. A formulation composed of different percentages of lipids and surfactant compositions, and cholesterol (CH) has been tested to optimize the skin permeation of bacitracin zinc. The stability of vesicles characterized by drug encapsulation and vesicle size was also measured. The possible mechanisms for skin permeation of liposomes and niosomes were elucidated with the results of a series of skin permeation experiments.

 

MATERIALS AND METHODS:

Materials:

Bacitracin zinc (BZ) was purchased from Himedia Chemicals, (Mumbai, India). Dimyristoyl-L-α-phosphatidylcholine, egg phosphatidylcholine (Egg PC; 99%), cholesterol (CH) were purchased from Sigma Chemical Co. (USA). Sorbitan monopalmitate (Span 40) and sorbitan monostearate (Span 60) were purchased from Loba Chemie (Mumbai, India). All other chemicals and solvents were of analytical grade. Double distilled water was used throughout the study. Spargue-Dawely rats were used for all the animal studies.

 

Methods:

Preparation of Liposomes and Niosomes:

Liposomes and niosomes were prepared using thin film hydration method (Agarwal et al., 2001). Accurately weighed quantities of the surfactant (Span 40; Span 60) and cholesterol in different molar ratios for niosomes, and phospholipids for liposomes, were dissolved in 9 ml of a chloroform/methanol mixture (2:1, v/v) in a round-bottom flask. 1% w/v bacitracin zinc dissolved in 5mL acetone: methanol mixture (4:1) was added to the lipid solution. The organic solvents were removed under vacuum in a rotary evaporator (Buchi evaporator, Switzerland) at 60°C to form a thin film on the wall of the flask. After removal of the last trace of organic solvent, the film was hydrated with 10 ml of phosphate buffered saline (PBS, pH 7.4) at 60°C for 1 h. The resulting niosomal or liposomal suspension was mechanically shaken for 1 h using a horizontal mechanical shaker (Kotterman, Hanigsen, Germany) at 60 rpm and 40°C leading to the formation of niosomes and liposomes respectively. The niosomal and liposomal suspension was left to mature overnight at 4°C.

 

Determination of Bacitracin zinc Encapsulation Percentage:

The bacitracin zinc containing vesicles were separated from the unencapsulated drug by filtering the dispersion under vacuum through a 0.05 mm filter (Millipore Co., USA); these vesicles were then washed with buffer to completely remove the free drug. The amount of entrapped bacitracin zinc was determined by lysis of the vesicles with absolute ethanol. The vesicle dispersion was mixed with an equal volume of ethanol and covered well with paraffin to prevent evaporation. The solution then was sonicated for 10 min in bath type sonicator to obtain clear solution (Law et al., 1994) and analyzed by high- performance liquid chromatography (HPLC) assay (Pavli and Kmetec, 2004). The entrapment efficiency was calculated using following formula.

Bacitracin zinc entrapment efficiency (%) =  

Amount of BZ entrapped      x 100

                        Total amount of BZ

 

 

Determination of Vesicle Size:

The vesicle sizes of the prepared liposome and niosome formulations were determined by light scattering based on laser diffraction using the Malvern Mastersizer (Malvern Instruments Ltd., Worcestershire, UK).

 

In vitro Permeation Study:

The permeation of bacitracin zinc from liposomes and niosomes was determined by using a Franz vertical diffusion cell. Skin of Spargue-Dawely rats (6 weeks old) was mounted on the receptor compartment with the stratum corneum (SC) side facing upward in-to the donor compartment. The donor compartment was filled with 2 ml liposome or niosome formulation with 1% (w/v) bacitracin zinc. A 10 ml aliquot of citrate - phosphate buffer (pH 7.4) was used as receptor medium. The available diffusion area of the cell was 1 cm2. The receptor compartment was maintained at 37°C, with magnetic stirring at 600 rpm. At appropriate intervals, aliquots of receptor medium were withdrawn and immediately replenished with an equal volume of fresh receptor solution. The samples from the receptor medium were analyzed by HPLC.

 

Stability Studies:

Based on in vitro characterization the formulations were selected for stability studies. The formulations were stored in glass tubes covered with aluminum foil at 30 ± 2°C for 2 months and characterized for vesicle size and drug content. (Devaraj et al., 2002)

 

Statistical Analysis:

Statistical significance of all the data generated was tested by analysis of variance (ANOVA) followed by student’s t-test. A confidence limit of p<0.05 was fixed for interpretation of the results using the software PRISM (Graphpad, San Diego, CA).

 

RESULTS AND DISCUSSION:

Determination of Entrapment Efficiency:

To obtain the highest encapsulation efficiency, several factors, including the inclusion of cholesterol, the structure of the surfactant, were investigated and optimized. PC with saturated long alkyl chains provided rigid bilayers with low permeability for encapsulated molecules (Talsma and Crommelin, 1993). The PC extracted from biological sources, such as egg yolk (Egg PC), was composed of a mixture of saturated and unsaturated fatty acids. Hence, the bacitracin zinc encapsulation of Egg PC liposomes was lower, as compared to DMPC liposomes is presented in Table 1.

 


Table1. Composition and Characterization of Bacitracin Zinc Liposomes and Niosomes (mean ± s.d., n=3).

Sr. No.

Code

Composition

Vesicle Size, nm

% Entrapment

1

DMPC

DMPC

1117 ± 29

63.28 ± 2.65

2

DMPC/CH

DMPC: CH = 7:3 (Molar Ratio)

1245 ± 37

84.41 ± 9.37

3

Egg PC

Egg PC: CH = 7:3(WeightRatio)

1162 ± 58

54.19 ± 11.36

Egg PC: CH = 5:5(Weight ratio)

1124 ± 76

38.66 ± 8.58

4

Span 40

Span 40: CH = 7:3(Molar Ratio)

956 ± 13

75.88 ± 6.53

Span 40: CH = 5:5 (MolarRatio)

1080 ± 90

58.32 ± 7.46

5

Span 60

Span 60: CH = 7:3 (MolarRatio)

1026 ± 34

79.20 ± 3.95

Span 60: CH = 5:5 (MolarRatio)

1189 ± 91

64.45 ± 4.27

 

 


Incorporation of cholesterol into niosomes and liposomes was found to increase the encapsulation efficiency of bacitracin zinc up to an optimum concentration of cholesterol. These results can be explained by the fact that an increase in cholesterol content resulted in an increase of micro viscosity of the membrane indicating more rigidity of the bilayers (Manosroi et al., 2003). Cholesterol has the ability to cement the leaking space in the bilayer membranes (Agarwal et al., 2001). It is obvious that further increase of cholesterol content, for niosomes and liposomes reduced the entrapment efficiency this could be due to the fact that cholesterol beyond a certain level starts disrupting the regular structure because of repulsive hydration force between bilayers leading to loss of drug entrapment (Betageri and Parsons, 1992). The data also reveals that the entrapment efficiencies for niosomes prepared using Span 60 were superior to those prepared using Span 40. This can be explained by many facts such as Span 60 has the highest phase transition temperature (Yoshioka et al., 1994). The length of alkyl chain of surfactant is a crucial factor in permeability. Long chain surfactant produces highest entrapment (Hao et al., 2002). Span 60 has a longer saturated alkyl chain (C16) compared to Span 40 (C14), so it produces niosomes with higher entrapment efficiency. The longer alkyl chain influences the HLB value of the surfactant mixture that by its turn directly influences the drug entrapment efficiency. The lower the HLB of the surfactant the higher will be the drug entrapment efficiency and stability as in the case of niosomes prepared using Span 60 (Raja Naresh et al., 1994).

 

Particle Size of Liposomes and Niosomes:

The vesicle size increased dramatically after adding CH in liposomes as shown in Table 1. This result might be explained in terms of the DMPC content. The addition of CH to liposomes reduced the DMPC content from 100% (DMPC liposomes) to 70% (DMPC/CH liposomes), inducing more aggregation because PC-enriched liposomes have difficulty aggregating due to the repulsive hydration force between bilayers (Hazemoto et al., 1993). Also, the results reveal that the niosomes prepared using Span 60 is larger in size than niosomes prepared using Span 40. Span 60 has a longer saturated alkyl chain compared to Span 40 as mentioned previously and it was reported that surfactants with longer alkyl chains generally gave larger vesicles (Manosroi et al., 2003). This would account for the highest entrapment efficiencies obtained with span 60 niosomes.

 

Skin Permeation of Bacitracin zinc Liposomes and Niosomes:

Cumulative amount–time profiles of free and liposomes or niosomes encapsulated bacitracin zinc across nude rat skin is given in Fig. 1. The curves were suitable to fit by first-order equation. DMPC liposomes reduced the bacitracin zinc permeation based on the total amount of bacitracin zinc permeated during the in vitro permeation study as shown in Table 2. The amount of permeated bacitracin zinc was increased in Egg PC liposomes compared to that in the free form or in DMPC liposomes, indicating the lipid composition of liposomes influenced the transdermal activity of entrapped bacitracin zinc. Egg PC contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylionsitol and unsaturated fatty acid. The presence of unsaturated fatty acids in the phospholipids may be responsible for the enhancer effect. Lecithin has a high affinity for SC. The packing nature of unsaturated fatty acids changed the fluidity of SC lipid structure and facilitated the skin permeation of drugs (Valenta et al., 2000; Valjakka-Koskela et al., 1998).

 

Table2. Total Amount of Bacitracin Zinc Permeated from Liposomes and Niosomes (µg/cm2) Across Intact Nude Mouse Skin during 24 ha (mean ± s.d., n=3).

Formulations

Total Amount Permeated during 24 hoursa (µg/cm2)

Free Drug

29.09 ± 3.30

DMPC

23.54 ± 1.82

DMPC:CH (7:3)

19.26 ± 1.30

Egg PC:CH (7:3)

49.14 ± 5.96

Egg PC:CH (5:5)

36.03 ± 3.44

Span 40:CH (7:3)

67.92 ± 4.07

Span 40:CH (5:5)

51.15 ± 2.55

Span 60:CH (7:3)

83.55 ± 6.05

Span 60:CH (5:5)

61.96 ± 2.13

a The data was calculated by the trapezoidal method from the area under curve (AUC) of flux-time profiles.

 

Transdermal permeation of bacitracin zinc of niosomes was much higher than that of free drug and liposomes as indicated by Fig. 1a and Fig. 1b. Surfactant in formulation always acts as a permeation enhancer, which might partly contribute, to the enhancement of bacitracin zinc permeation from niosomes. Another explanation was that non ionic surfactant fused at the interface of the SC, and the high local drug concentration in the bilayers generated a high thermodynamic activity of bacitracin zinc in the upper part of the SC (Sarpotdar and Zatz, 1986). Increasing the cholesterol molar ratio from 7:3 to 5:5 resulted in more intact lipid bilayers as a barrier for drug release and decreased its leakage by improving the fluidity of the bilayer membrane and reducing its permeability, which led to lower drug elution from the vesicles. These Results are in accordance with other reports (Manosroi et al., 2003). According to literature suggestions, vesicle size is not of great significance for the transport of hydrophilic drugs across skin as long as the diameter of vesicles is larger than 200 nm in size (Kristal and Sentjurc, 1999). It could be suggested that vesicles size does not play significant role (p>0.05) in the permeation of bacitracin zinc across skin. The comparative release data indicate that, by encapsulation of drug into liposomes and niosomes, it is possible to sustain and control the release of the drug for a longer duration (Ruckmani et al., 2000).

 

Fig. 1 In vitro cumulative amount time profiles of bacitracin zinc permeated across nude rat skin from liposomes (A)  and niosomes (B)

(A)               ○ = Free drug, □ = DMPC, ∆ = EggPC CH (7:3), ■= DMPC CH (7:3), ▲= Egg PC CH (5:5).

(B)               ○ = Free drug, □ = SPAN 40 CH (7:3), ■= SPAN 40 CH (5:5), ∆ = SPAN 60 CH (7:3), ▲= SPAN 60 CH (5:5).

 

Permeation of Bacitracin zinc Liposomes and Niosomes across Various Types of Skin:

Three types of interaction between the skin and vesicles may induce the enhancing effect on transdermal drug delivery: (1) adsorption and fusion of drug loaded vesicles onto the surface of the skin leads to a high thermodynamic activity gradient of the drug–SC interface; (2) the effect of vesicles on SC may cause changes in drug permeation kinetics due to an impaired barrier function of the SC for the drug (Schreier and Bouwstra, 1994; Touitou et al., 1994); (3) The lipid bilayers of niosomes and liposomes act as a rate-limiting membrane barrier for drugs.

 

In order to explore the mechanisms of bacitracin zinc vesicles across the skin, Egg PC liposomes and Span 60 niosomes were selected as model vesicles to investigate the possible reasons for bacitracin zinc permeation enhancement. Pretreatment of skin with phospholipid and non-ionic surfactant was performed to clarify whether phospholipid or surfactant affected the structure of skin. Egg PC or Span 60 pretreated the nude rat skin at the same concentration in vesicles (pH 7.4) buffer for 12 h, and then 1% w/v bacitracin zinc free drug (pH 7.4) buffer was applied to the skin for 24 h for in vitro permeation experiments. Fig. 2 shows that the bacitracin zinc permeation across Egg PC treated and span 60 treated skin was significantly higher (p<0.05) than that across non-treated skin. One of the possible mechanisms for enhancement of the permeability of bacitracin zinc is structure modification of the stratum corneum. It has been reported that the intercellular lipid barrier in the stratum corneum would be dramatically looser and more permeable following treatment with liposomes and niosomes (B.W. Barry, 2001; Ogiso et al., 1996). Both phospholipids and nonionic surfactants can act as penetration enhancers, which are useful for increasing the permeation of many drugs. Fusion of niosome vesicles to the surface of skin, demonstrated in a previous report (B.W. Barry, 2001), results in higher flux of the drug due to direct transfer of drug from vesicles to the skin. From the results, Egg PC and Span 60 could serve as permeation enhancers for bacitracin zinc delivery via the skin.

 

Figure2. In vitro cumulative amount time profiles of bacitracin zinc permeated across optimized liposomes and niosomes pretreated nude rat skin.

○ = Free drug, ● = EggPC CH (7:3) pretreatment, ∆ = SPAN 60 CH (7:3) pretreatment.


Table3. Total Amount of Bacitracin Zinc Permeated from Liposomes and Niosomes (µg/cm2) Across Various Types of Skin during 24 ha (mean ± s.d., n=3).

Formulations

Free Drug

Egg PC: CH (7:3)

Span 60:CH (7:3)

Across intact skin

29.09 ± 3.30

49.14 ± 5.96

83.55 ± 6.05

Across pretreated skin

-

75.31 ± 5.98

188.92       ± 8.76

Across stripped skin

216.73 ± 8.44

235.10 ± 7.28

200.21 ± 10.81

Direct addition (physical mixture)

-

34.570 ± 3.83

46.47          ± 8.21

The data was calculated by the trapezoidal method from the area under curve (AUC) of flux-time profiles.

 

Table4. Stability of Selected Bacitracin Zinc Liposomes and Niosomes at 30 ± 2°C Expressed as Vesicle Size and Encapsulation Efficiency (mean ± s.d., n=3).

Formulation

Vesicle Size, nm

Encapsulation Efficiency

Initial

30 Days

60 Days

Initial

30 Days

60 Days

DMPC

1117 ± 29

1149 ± 34

1191 ± 63

63.28 ± 9.37

47.73 ± 7.85

25.06 ± 3.65

DMPC:CH

1245 ± 37

1309 ± 51

1374 ± 54

84.41 ± 2.65

62.16 ± 3.14

50.27 ± 12.74

Egg PC:CH (7:3)

1162 ± 58

1219 ± 25

1282 ± 61

54.19 ±11.36

45.59 ± 5.84

40.76 ± 3.91

Span 40:CH (7:3)

956 ± 26

988 ± 23

1013 ± 38

75.88 ± 6.53

66.06 ± 5.11

59.44 ± 7.28

Span 60:CH (7:3)

1026 ± 34

1067 ± 43

1105 ± 53

79.20 ± 3.95

72.34 ± 4.62

68.97 ± 3.49

 


The permeation of bacitracin zinc across SC-stripped skin was measured using Egg PC liposomes and Span 60 niosomes to clarify the degree of the vesicle contribution to permeation across the SC as shown in Fig. 3. The total bacitracin zinc amount permeated across the stripped skin was much higher (p<0.05) than that across intact skin in the free drug form implicating the barrier function of SC for the skin permeation of the drug as presented in Table 3. Bacitracin zinc in liposomes and niosomes also permeated more rapidly across the stripped skin than across the intact skin. However, the enhancement ratio (ER=total amount permeated stripped skin/total amount permeated intact skin) of liposomes (ER=4.78) and niosomes (ER=2.40) were smaller as compared with that of the free drug form (ER=7.45). The action of liposomes and niosomes as permeation enhancers might predominantly be on the intercellular lipids of SC, raising the fluidity and weakness of the SC (Vora et al., 1998; Valjakka-Koskela et al., 1998). The enhancement ratio after stripping was lower for niosomes than for liposomes. This result might suggest a weaker SC barrier effect when niosomes were applied. Additionally, the result suggests that liposomes and niosomes largely contribute to the rapid permeation of bacitracin zinc across the SC, which may be due to the higher diffusion of vesicles with drug in the SC. These results are consistent with the reports (Ogiso et al., 1996).

Bacitracin zinc was formulated as suspensions containing the same amounts of Egg PC liposomes and Span 60 niosomes, without any further preparation procedure. This physical mixture, by directly adding all the components in the donor, was used to study the effects of liposomes or niosomes vesicles on the permeation of bacitracin zinc as shown in Fig. 4. It was clearly demonstrated that the Span 60 niosomes and Egg PC liposomes has significantly higher permeation than Span 60 physical mixture and Egg PC physical mixture respectively. Thus, the permeation of bacitracin zinc from the formulations containing the physical mixture cannot reach to the extent from niosomes and liposomes. It is suggested that factors other than the permeation enhancer effect of phospholipids or surfactants in the vesicles are involved in the enhancement of bacitracin zinc permeation across skin. Hence a higher amount of total drug may be delivered across the skin, directly via vesicles, relative to simple aqueous solutions. Based on the previously proposed mechanisms of penetration enhancement with the niosomal and liposomal system through skin membrane (Manconi et al., 2001; Shahiwala and Misra, 2002; Vora et al., 1998; B.W. Barry, 2001), and experimental data and theoretical analysis support the concept that the direct transfer of drug from liposome and niosome to skin occurs only when the drug is intercalated within the bilayers. Phospholipids are known to have a high affinity for biological membranes. It is shown that mixing of liposomes with the skin lipids in the intercellular layers could be one mechanism contributing to the enhancement of drug permeation of the skin to lipid vesicles (Ogiso et al., 1996).

 

Fig.3. In vitro cumulative amount time profiles of bacitracin zinc permeated across intact and stratum corneum stripped skin from optimized liposomes and niosomes.

○ = Free drug across stripped skin, □ = EggPC CH (7:3) across stripped skin, ∆ = SPAN 60 CH (7:3) across stripped skin, ● = Free drug across intact skin,■= EggPC CH (7:3) across intact skin,▲= SPAN 60 CH (7:3) across intact skin.

 

Stability of bacitracin zinc liposomes and niosomes:

stability data of liposomes and niosomes is given in Table 4. The encapsulated drug tends to leak out from the bilayer structures during storage. A significant loss in bacitracin zinc encapsulation of DMPC liposomes was noted after incubation in suspension form for 2 months. Encapsulation loss was always associated with an increase in vesicle size indicating that complete swelling of bilayer and hence formation of more uniform vesicles upon storage.

Phospholipid loss, in the presence of water, from the liposome bilayers leads to the formation of pores and leakage. After incorporation of CH, leakage of bacitracin zinc is significantly reduced. Inclusion of CH in liposomes improved the fluidity of the bilayer membrane and reduced the permeability of drugs through the membrane. (Manosroi et al., 2003; Vemuri et al., 1990). This effect lessened the leakage of the entrapped drug from the liposomes and resulted in a higher stability of the formulation. Both Span 40 and Span 60 niosomes showed good stability according to encapsulation and vesicle size. The problems of phospholipid hydrolysis and fatty acid peroxidation in liposomes were not observed for non-ionic surfactants.

 

Figure4. In vitro cumulative amount time profiles of bacitracin zinc permeated across nude rat skin from vesicles or physical mixtures.

○ = Free drug, □ = Egg PC CH liposome, ∆ = SPAN 60 CH niosome, ■= EggPC CH (7:3) physical mixture, ▲= SPAN 60 CH (7:3) physical mixture.

 

CONCLUSION:

Topical delivery of bacitracin zinc was developed and investigated for the liposome/niosome system. The results show that the optimized niosome prepared in this study has a better skin permeation potential, sustained release characteristic and higher stability compared to liposomes. The ability of liposomes and niosomes to modulate drug delivery makes the two vesicles useful to formulate topical bacitracin zinc.

 

ACKNOWLEDGEMENTS:

The authors would like to acknowledge the industries provided free samples and NDMVP Samaj’s, College of Pharmacy, Nasik for providing necessary research facilities.

 

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Received on 08.11.2009       Modified on 03.02.2010

Accepted on 07.04.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1295-1300