4. Surfactants used in the preparation of Niosomes not require any special handling and storage conditions.

5. Niosomes are having greater flexibility with respect to composition, fluidity and size. They can be designed based on desired situation.

6. Niosomes can be administered by oral, parenteral and topical routes.

7. Niosomes can improve skin penetration; enhance oral bioavailability of poorly absorbed drugs.

8. The nonionic surfactants used in Niosomes are biodegradable, biocompatible and non immunogenic.

9. Therapeutic performance of drug can be improved by targeting, delayed clearance from circulation.

Liposomes Vs Niosomes²

Table1: Comparision between Liposomes and Niosomes

s.no.	Liposomes	Niosomes
1.	Liposomes undergo degradation by hydrolysis or oxidation during storage.	Degradation by hydrolysis or oxidation not observed during storage.
2.	Phospholipids used in the preparation of liposomes are suffering from various purity problems.	Phospholipids are not used in the preparation of Niosomes.
3.	liposomes are prepared from double-chain phospholipids	prepared from uncharged single-chain surfactant

Thecomponents present in the Niosomes are non ionic surfactants, cholesterol, water and drug.³

The association of non-ionic surfactant monomers into vesicles on hydration is due to two opposing forces ⁴

1. Interfacial tension between water and the hydrocarbon portion of the amphiphile.

2. The steric, hydrophilic and/or ionic repulsion between the head groups.

These two opposing forces result in a molecular assembly.

Composition Considerations in Niosomes

Various non ionic surfactants used in the preparation of Niosomes are ⁴Sorbitan monostearate (span 60), Polyoxyethylene 2 stearylether (brij 72), Polyoxyethylene sorbitan monostearate (tween 61), Glyceryl monostearate , Polyoxyethylene 4 laurylether (brij 30) , Di glyceryl monolaurate , Tetra glyceryl monolaurate

The mean size of Niosomes increases regularly with increase in the HLB from Span 85(1.8) to span 20(8.6). Vesicles obtained from the long alkyl chain (C18) surfactants give higher entrapment efficiency and were more stable than the shorter alkyl chain (C12) surfactants.

The effect of Niosome forming surfactant ⁴

Increased hydrophilicity causes the increased leakage of low molecular weight drugs from aqueous compartments, decreased stability of Niosome preparation and improved transdermal delivery of hydrophobic molecules. Whereas increased hydrophobicity causes the decreased leakage of low molecular weight drugs from aqueous compartments, increased encapsulation, increased stability of Niosome preparation and decreased toxicity.

Role of cholesterol

Inclusion of cholesterol in Niosomes increases its diameter and entrapment efficiency. Presence of cholesterol in bilayer reduces permeability and improves retention of solute. 1:1 molar ratio of the surfactants to cholesterol is generally used.³

Methods of Preparation of Niosomes^2,5
Niosomes can be prepared by a number of methods which are as follows:

(a) Ether injection method

In this method, the surfactant solution is prepared by dissolving it in diethyl ether. The above solution is then introduced using an injection (14 gauge needle) into warm water or aqueous media containing the drug maintained at 60°C. Evaporation of the ether leads to the formation of single layered vesicles. The particle size of the Niosomes formed depend on the conditions used, and can range anywhere between 50-1000µm.

(b) Hand shaking method (Thin Film Hydration Technique)

In this method a mixture of the surfactant and cholesterol are dissolved in a volatile organic solvent such as diethyl ether or chloroform in a round bottom flask. The organic solvent is removed at room temperature using a rotary evaporator, which leaves a thin film on the walls of the flask. This dried surfactant film can then be rehydrated with the aqueous phase, to yield multilamellar Niosomes. The multilamellar vesicles thus formed can further be processed to yield unilamellar Niosomes using microfluidization, sonication or membrane extrusion techniques.

(c) Sonication Method

Aqueous phase was added to the surfactant/cholesterol mixture and the mixture was probe sonicated at 60° C for 3 minutes to produce niosomes. 5,6 carboxy fluorescein is entrapped niosomes by sonication method.

(d) Microfludisation

This is a recent technique to prepare small MLVS. A Microfludizer is used to pump the fluid at a very high pressure (10,000psi) through a 5 pm screen. Thereafter; it is forced along defined micro channels, which direct two streams of fluid to collide together at right angles, thereby affecting a very efficient transfer of energy. The lipids can be introduced into the fluidizer. The fluid collected can be recycled through the pump until vesicles of spherical dimensions are obtained. This results in greater uniformity, small size and better reproducible niosomes.

(e) Multiple membrane extrusion method

Mixture of surfactant, cholesterol and dicetyl phosphate in chloroform is made into thin film by evaporation. The film is hydrated with aqueous drug polycarbonate membranes, solution and the resultant suspension extruded through which are placed in series for upto 8 passages. It is a good method for controlling niosome size.

(f) Reverse phase evaporation technique

In this method cholesterol and surfactant (1:1 ratio) solution is prepared using a mixture of ether and chloroform. Drug in aqueous phase is added to this, and the resulting two phases are sonicated at 4-5°C. A clear gel is formed which is further sonicated after the addition of phosphate buffered saline (PBS). Later the temperature is raised to 40°C and pressure is reduced to remove the organic phase. This results in a viscous Niosome suspension which can be diluted with phosphate buffered saline and heated on a water bath at 60°C for 10 mins to yield Niosomes.

(g) Trans membrane pH gradient Drug Uptake Process

In this method, a solution of cholesterol and surfactant is made in chloroform. The solvent is evaporated under reduced pressure to get a thin film on the wall of the round bottom flask. This film is then hydrated using citric acid solution (300mM, pH 4.0) by vortex mixing. The resulting multilamellar vesicles are then treated to three freeze thaw cycles and sonicated. To the suspension, aqueous solution containing drug is added and vortexed. The pH of the sample is then raised to 7.0-7.2 using 1M disodium phosphate (this causes the drug which is outside the vesicle to become non-ionic and can then cross the niosomal membrane, and once inside it is again ionized thus not allowing it to exit the vesicle). Later the mixture is heated at 60°C for 10 minutes to give Niosomes.

(h) The Bubble Method

It is a technique which allows the preparation of Niosomes without the use of organic solvents. The bubbling unit consists of a round bottom flask with three necks, and this is kept in a water bath to control the temperature. Water-cooled reflux and thermometer is positioned in the first and second neck respectively, while the third neck is used to supply nitrogen. Cholesterol and surfactant are dispersed together in a buffer (pH 7.4) at 70°C. This dispersion is mixed for 15 seconds with high shear homogenizer and immediately afterwards, it is bubbled at 70°C using the nitrogen gas to yield Niosomes.

(i) Formation of Niosomes from Proniosomes

To create proniosomes, a water soluble carrier such as sorbitol is first coated with the surfactant. The solution of the surfactant with cholesterol in a volatile organic solvent is sprayed onto the powder of sorbitol kept in a rotary evaporator. The evaporation of the organic solvent yields a thin coat on the sorbitol particles. The resulting coating is a dry formulation in which a water soluble particle is coated with a thin film of dry surfactant. This preparation is termed Proniosome. The Niosomes can be prepared from the proniosomes by adding the aqueous phase with the drug to the proniosomes with gentle agitation at a temperature greater than the mean transition phase temperature of the surfactant.

Table 2: List of drugs incorporated by various methods into Niosomes

S. No	Method	Drugs
1	Ether injection method	sodium stibogluconate⁶
2	Hand shaking method	Methotrexate⁷, Doxorubicin⁸
3	Sonication	9-desglycinamide8-arginine vasopressin⁹, Estradiol¹⁰
4	Reverse phase evaporation technique	5-Fluorouracil¹¹
5	Multiple membrane extrusion method	Tretinoin¹²
6	Trans membrane pH gradient Drug Uptake Process	Timolol Maleate¹³
7	Formation of Niosomes from ProNiosomes	Levonorgestrol¹⁴

Characterization of Niosomes⁵

Various characterization tests performed for Niosomes are

Entrapment efficiency

The drug entrapped in Niosomes is determined by complete vesicle disruption using 50% n-propanol or 0.1% Triton X-100 and analysing the resultant solution by appropriate assay method for the drug. Where,

Entrapment efficiency (EF) = (Amount entrapped /total amount) x 100

Vesicle diameter

Niosomes, assume spherical shape and so their diameter can be determined using light microscopy, photon correlation microscopy and freeze fracture electron microscopy. Freeze thawing¹⁵(keeping vesicles suspension at –20°C for 24 hrs and then heating to ambient temperature) of Niosomes increases the vesicle diameter, which might be attributed to fusion of vesicles during the cycle.

In-vitro release

A method of in-vitro release rate study is performed with the use of dialysis tubing. A dialysis sac is washed and soaked in distilled water. The vesicle suspension is poured into a bag made up of the tubing and sealed. The dialysis sac containing the vesicles is placed in 200 ml of buffer solution in a 250 ml beaker with constant shaking at 37°C. At various time intervals, the buffer is analyzed for the drug content by an appropriate assay method¹⁶.

Niosome Applications^17,18,19

Several Niosome delivery applications are

Drug targeting

Niosomes are widely used in targeted drug delivery.

(a) Anti cancer drugs

Methotrexate

Niosome formulations of methotrexate containing 47.5 or 30% cholesterol have higher serum levels when administerd in the form of solution.²⁰ Tumour bearing mice administered with methotrexate Niosomes formulated with Span 60 through intravenous route showed 23 fold increase in the area under the plasma level time curve.²¹ When methotrexate Niosomes formulated with Span 60 were administered following macrophage activation with muramyl dipeptide –gelatin conjugates and methotrexate tumoricidal activity was also increased.²¹Administration of 8 mm Span 85 methotrxate nisomes intraperitoneally resulted in targeting of metastitial cancer of lymphatic system.²²

Doxorubicin¹⁷

Sorbitan monostearate poly oxy ethylene coated Niosomes circulate for prolonged periods. It’s area under the plasma level time curve is increased 6 fold when compared with it’s solution form, concentration of doxorubicin at tumour increased by 50% and tumorocidal activity was doubled. The long circulation time was due to poly oxy ethylene coating,which prevents particle recognition and the uptake by the liver and spleen.

(b) Antiinfectives

In case of Leishmaniasis parasite invades the cells of liver and spleen¹⁸. Most commonly used drug is antimonials, which are related to arsenic. Arsenic at high concentration damages the liver, heart and kidney. Liver targeting can be achieved with hexadecyl triglycerol sodium stibogluconate Niosomes. The anti-parasitic activity of sodium stibogluconate is increased 10 fold by encapsulation into Niosomes¹⁷.

Anti tuberculosis agent, Rifampicin encapsulated with in Span 85 based Niosomes found to accumulate in the lung of mice.²³ Diclofenac encapsulated in Niosomes formulated with polysorbate 60 were found to reduce the inflammation in rats to a greater extent than free drug.²⁴

(c) Delivery to the brain^17,18

Delivery of peptides across blood brain barrier is a great challenge. However , glucose coated Niosomes able to achieve brain delivery of hydrophilic peptides. These vesicles uses the glucose transporter at the blood brain barrier, possibly by initially concentrating drug at the barrier, and have been shown to deliver intact vasoactive intestinal peptide to the posterior and anterior parts of the brain.

Topical use of Niosomes

(i) Transdermal

Several mechanisms were proposed to modulate drug transfer across the skin. They are

(a). By Adsorption and fusion of Niosomes onto the skin surface.

(b). Niosomes can act as penetration enhancers.

Topical administration of Flurbiprofen Niosomes formulated with Span 60 in a hydroxy propyl methyl cellulose base gave high area under plasma level time curve when compared with flurbiprofen suspension.²⁵

Transdermal delivery is specifically enhanced when hydrophilic surfactants such as poly oxy ethylene-7-dodecyl ether or poly oxy ethylene -8-lauryl ester are used to produce elastic vesicles.

(ii). Ocular

Cyclopentolate encapsulated within polysorbate 20 Niosomes were found to penetrate the cornea. There was increased mydriatic response with niosomal formulation irrespective of the P^Hof the formulation.²⁶ For the topical treatment of glaucoma, niosomal formulations have emerged in the form of Carbopol 934p coated sorbitan monostearate acetazolamide Niosomes , both chitosan and carbopol 934p coated sorbitan monostearate Timolol maleate Niosomes and sorbitan monopalmitate Timolol maleate discomes.

Niosomal vaccines

Encapsulation of both antigens and DNA encoding for antigens in Niosomes results in the stimulation of humoral and cellular immune response to the antigens. Surfactants have immunostimulatory properties.¹⁷Increased protection against an infectious challenge has been observed in mice that are vaccinated against herpes simplex virus type I, vesicles prepared from synthetic amphipiles.¹⁸

Niosomes as imagining agents

Apart from the use of Niosomes as drug carrier they are also used as diagnostic agents. Intravenous administration of C₁₆G₃ and C₁₆C₁₂G₇ Niosomes containing cholesterol and stearylamine encapsulating the radioopaque agent iopromide were concentrated in kidneys.²⁷This kidney targeting may be due to the presence of positive charge on the Niosome surface although no neutral controle was used in the study. The highest kidney concentration was observed in C₁₆G₃ Niosomes compared with C₁₆C₁₂G₇ Niosomes. It may be due to formation of less fluid bilayer by C₁₆G₃ Niosomes. It may be due to formation of less fluid bilayer by C₁₆G₃Niosomes.

Table3: Examples of niosomal delivery applications ¹⁷

S.No	Area	Formulation and route of administration	Advantages
1.	Cancer	Sorbitan monostearate doxorubicin Niosomes – IV hexadecyl triglycerol ether doxorubicin Niosomes – IV sorbitan monostearate methotrexate Niosome - IV	Tumor drug levels were increased by 50% Increase in tumor drug levels compared to solution form. Increase in plasma levels by 23 times compared to drug in solution form.
2.	Glaucoma	Timolol maleate sorbitan monostearate Niosomes coated with chitosan -topical	Intraocular pressure will be reduced at sustained rate. This is twice active than a commercial gel.
3.	Anti – infectives	hexadecyl triglycerol ether Niosomes - IV	Antimony levels are twice in liver incase of Niosomes compared with solution.
4.	Neurological disorders	N-palmitoyl glucosamine vasoactive intestinal peptide (VIP) Niosomes.- IV	VIP will be delivered to brain when encapsulated in Niosomes, but not if administered as drug in solution.
5.	vaccination	palmitoyl glycerol herpes simplex virus type 1 Niosomes- IM	Neutralizing antibodies are higher in Niosomes than antigen in phosphate buffered saline. Survival rate is higher when challenged with virus.
6.	Diagnostic imagining agents	N-palmitoyl glucosamine gadolinium Niosomes and n-palmitoyl glucosamine gadolinium Niosomes coated with poly (oxyethylene)- IV	Higher tumor (PC3 tumor cells) to muscle ratio of contrast agent 24 h after dosing with glucose or poly(oxyethylene) bearing Niosomes when compared to sorbitan monostearate Niosome

Other applications²

(a) Sustained release

Liver act as a depot for methotrexate after Niosomes are takenup by the liver cells.²⁰ In case of drugs with low therapeutic index and low water solubility sustained release can be achieved with Niosomes.

(b) Localised drug action

Localised drug action of Niosomes is due to it’s size and low penetrability through epithelium and connective tissue. Localized drug action enhances the potency and reduces the side effects. E.g.Antimonials when taken up by the mononuclear cells resulting in localization of drug, increase in potency and decrease in dose and toxicity.

STABILITY AND TOXICITY OF NIOSOMES

Compared to liposomes, niosomes are more stable. Surfactants are used in the preparation of niosomes, which may be a cause of toxicity. However, there are no reports available on the in vivo toxicity of niosomes linked with the concentration of ester or ethers surfactants used in the preparation of vesicles.

CONCLUSION:

Nanovesicles, especially Niosomes are interesting delivery systems for pharmaceuticals and cosmetics. Topically applied Niosomes can increase the residence time of drugs or cosmetic chemicals in the SC and epidermis and reduce the systemic absorption of the drugs or cosmetic chemicals. However, elastic Niosomes have soft and flexible vesicular characteristics. These properties allow them to penetrate easily into deeper layers of the skin and circulation. The optimized developed elastic niosomal vesicular formulations by adjusting their compositions and sizes can be promising means for not only cosmeceutical applications, but also the topical non-invasive treatment of local and systemic disorder of many pharmaceuticals as well.

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Received on 08.07.2010

Accepted on 02.08.2010

Research Journal of Pharmaceutical Dosage Forms and Technology. 3(2): March-April 2011, 42-47