Niosomes: A promising drug delivery system
Introduction In the era of novel drug delivery system (NDDS) emphasis is given on spatial placement of drug for chronic conditions. Targeted delivery of anticancer and anti-infective drugs appears to be challenging but achievable task with the use of novel drug delivery systems. Different novel approaches used for delivering these drugs include liposomes, microspheres, nanotechnology, micro-emulsion, antibody-loaded drug delivery, magnetic microcapsules, implantable pumps and niosomes . In 1975 it appeared that non-ionic surfactants that had been produced on an industrial scale for several years might be appropriate components for vesicles . Non-ionic surfactant based vesicles (niosomes) are formed from the self-assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer structures . The process vesicle formulation by self-assembly of nonionic surfactants is rarely spontaneous and usually requires some input of energy through physical agitation, extrusion or heat . These non-ionic surfactant vesicles appear to be similar to liposomes in terms of their physical properties, structures and methods of preparation . On the other hand, niosomes have more advantages over the liposomes, such as entrapment of more substances, higher stability, needlessness of handling or storing in special conditions, and the availability as well as inexpensiveness of prepared materials . Niosomes enhances the penetration through the skin and the bioavailability of many drugs like dicolfenac diethylammonium , enhance penetration of peptide drugs , improve oral bioavailability of paclitaxel , griseofulvin . Factors influence the formation of niosomes Nature of surfactants Theoretically niosomes formation requires the presence of a particular class of amphiphile and aqueous solvent. In certain cases cholesterol is required in the formulation and vesicle aggregation for example may be prevented by the inclusion of molecules that stabilise the system against the formation of aggregates by repulsive steric or electrostatic effects. Such amphiphiles by definition must possess a hydrophilic head group and a hydrophobic tail. The hydrophobic moiety may consist of one or two alkyl or perfluoroalkyl groups or in certain cases a single steroidal group. The alkyl group chain length is usually from C12-C18 . The ether type surfactants with single chain alkyl as hydrophobic tail is more toxic than the corresponding dialkylether chain while the ester type surfactants are chemically less stable than ether type surfactants and the former is less toxic than the latter due to the ester-linked surfactant degraded by esterases to triglycerides and fatty acid in vivo . Surfactants such as C16EO5 (poly-oxyethylene cetyl ether) or C18EO5 (poly-oxyethylene steryl ether) are used for preparation of polyhedral vesicles . Structure of surfactants The geometry of vesicle to be formed from surfactants is affected by its structure, which is related to critical packing parameters. On the basis of critical packing parameters of the surfactants can predicate geometry of vesicle to be formed. Critical packing parameters can be defined using following equations  CPP (Critical Packing Parameters) = V/(Ic X a0) Where v = hydrophobic group volume. Ic= the critical hydrophobic group length. a0= the area of hydrophilic head group. as given below, If CPP 1 formation inverted micelles. Membrane Composition The stable niosomes can be prepared with addition of different additives along with surfactant and drugs. Niosomes formed have a number of morphologies and their permeability and stability properties can be altered by manipulating membrane characteristics by different additives. In case of polyhedral niosomes formed from C16G2, the shape of these polyhedral niosomes remains unaffected by adding low amount of solulan C24 (cholesteryl poly-24-oxyethylene ether), which prevents aggregation due to development of steric hindrance. In contrast spherical niosomes are formed by C16G2: cholesterol: solulan (49:49:2). The mean size was found to be influenced by membrane composition . Nature of encapsulated drug Entrapment of drug in niosomes increases vesicle size, probably by interaction of solute with surfactant head groups, increasing the charge and mutual repulsion of the surfactant bilayers, thereby increasing vesicle size. In polyoxyethylene glycol (PEG) coated vesicles; some drug is entrapped in the long PEG chains, thus reducing the tendency to increase the size. The hydrophilic lipophilic balance of the drug affects degree of entrapment [8, 13] Temperature of hydration The shape and size of the niosomes were affected directly by the hydration temperature. The ideal temperature for hydration is preferred to be above the gel to liquid phase transition temperature of the system. Temperature change of the niosomal system affects assembly surfactants into vesicles and also induces vesicle shape transformation . Characterization of niosomes Size Shape of niosomes vesicles assumed to be spherical, their mean diameter can be determined by using laser light scattering method. Generally the particle size distribution of niosomes is between 10 - 150 nm. Niosomes exhibit different morphologies and size depending on the type of nonionic surfactants and lipids. Discoid and ellipsoid vesicles (~ 60 um in diameter) with entrapped aqueous solutes are formed when hexadecyl diglycerol ether is solubilized by Solulan C24 [cholesteryl-poly(24-oxyethylene ether)]. Polyhedral niosomes are formed when cholesterol content is low in the same system. Polyhedral niosomes are thermoresponsive and release the encapsulated drug when heated above 350C. This can be useful for sunscreen formulations in which the sunscreen can be released on exposure to sun. Niosomes have been shown to penetrate the skin and enhance the permeation of drugs. Span niosomes showed significantly higher skin permeation and partitioning of enoxacin than those shown by liposomes and the free drug. Niosomes (100 nm) are mainly localized in the SC, but some can penetrate deeper layers of the skin. The niosomes dissociate and form loosely bound aggregates, which then penetrate to the deeper strata. Furthermore, the skin penetration has been attributed to the flexibility of niosomes, and this is supported by the fact that a decrease in cholesterol content increases the drug penetration through the skin. Furthermore, the nonionic surfactant can also modify the intercellular lipid structure in the SC to enhance skin permeability. In addition, adsorption and fusion of niosomes with the skin surface increase the drug's thermodynamic activity, leading to enhanced skin penetration. In vitro studies have found that the chain length of alkyl polyoxyethylene in niosomes did not affect the cell proliferation of human keratinocytes, but ester bond was found to be more toxic than ether bond in the surfactants. Entrapment efficiency (EE) The entrapment efficiency is expressed as: EE% = (Amount of Drug Entrapped/Total Amount Added)X100 The entrapment efficiency is affected by the following factors: * Surfactants: The chain length and hydrophilic head of non-ionic surfactants affect entrapment efficiency, such as stearyl chain C18 non-ionic surfactant vesicles show higher entrapment efficiency than lauryl chain C12 non-ionic surfactant vesicles. The tween series surfactants bearing a long alkyl chain and a large hydrophilic moiety in the combination with cholesterol at 1:1 ratio have highest entrapment efficiency for watersoluble drugs . * Cholesterol contents: The incorporation of cholesterol into bilayer composition of niosome induces membrane-stabilizing activity and decreases the leakiness of membrane. Hence, incorporation of cholesterol into bilayer increases entrapment efficiency. The permeability of vesicle bilayer to 5, 6-carboxy flourescein (CF) is reduced by 10 times due to incorporation of cholesterol  Preparation of niosomes Preparation of niosomes generally involves three steps: 1. Hydration of the surfactant/lipid mixture at elevated temperature. 2. Size reduction of niosomes. 3. Remove of the unentrapped drug. To achieve these aforementioned steps different techniques and methods can be used. The hydration step can be done using the following methods: 1. The injection of an organic solution of surfactants: lipids in an aqueous solution of the drug to be encapsulated which is heated above the boiling point of the organic solvent (ether injection). 2. The formation of a surfactant:lipid film by the evaporation of an organic solution of surfactants: lipids. This film is then hydrated with a solution of the drug (hand shaking) [5, 16]. 3. The formation of an oil in water (o:w) emulsion from an organic solution of surfactants:lipids and an aqueous solution of the drug. The organic solvent is then evaporated to leave niosomes dispersed in the aqueous phase. In some cases, a gel results which must be further hydrated to yield niosomes. (reverse phase evaporation) 4. The injection of melted lipids:surfactants into a highly agitated heated aqueous phase in which presumably the drug is dissolved or the addition of a warmed aqueous phase dissolving the drug to a mixture of melted lipids and hydrophobic drug . 5. The addition of the warmed aqueous phase to a mixture of the solid lipids: surfactants . 6. Niosomes may also be formed from a mixed micellar solution by the use of enzymes. A mixed micellar solution of C16G2, DCP, polyoxyethylene cholesteryl sebacetate diester (PCSD) converts to a niosome dispersion when incubated with esterases. PCSD is cleaved by the esterases to yield polyoxyethylene, sebacic acid and cholesterol. Cholesterol in combination with C16G2 and DCP then yields C16G2 niosomes . 7. The homogenisation of a surfactant:lipid mixture followed by the bubbling of nitrogen gas through this mixture. Apparently the homogenisation step may be omitted from the procedure without affecting particle size, although a longer 'bubbling' time was required . 8. Preparation of niosomes from proniosomes a procedure is described for producing a dry product which may be hydrated immediately before use to yield aqueous niosome dispersions similar to those produced by more conventional methods . 9. Remote loading which relies on the presence of intravesicular trap such as ammonium sulphate or alternatively a pH gradient across the membrane. The use of ammonium sulphate gradients was first reported in the formation of doxorubicin liposomes and which is the same method used to prepare Caelyx(r). This method is also used for preparation of doxorubicin niosomes and is only suited to the formulation of amine drugs. The size reduction of niosomes can be done by applying the following techniques: 1. Probe sonication which yields C16G3 niosomes in the 100-140 nm size range [5, 16]. 2. Extrusion through 100 nm Nucleopore filters which yields sodium stibogluconate C16G3 niosomes in the 140 nm size range. 3. In some instances the combination of sonication and filtration (220 nm Millipore(r) filter) has been used to achieve DOX loaded Span 60 niosomes in the 200 nm size range . 4. The achievement of sub-50 nm size is possible by use of a microfluidizer. 5. High pressure homogenisation also yields vesicles below 100 nm in diameter although drug loading is ultimately sacrificed to achieve this small size. 6. Laser diffraction is used to reduced niosomes size up to nano range. Removal of unentrapped drug Different methods and techniques used. The eluting of drug is achieved by using sephadex-50 in presence of phosphate buffer saline pH 7.4 as eluting fluid. Other methods like exhaustive dialysis, centrifugation and ultracentrifugation can also be used [8, 19-21]. Application of niosomes in drug delivery Delivery of anti-cancer drugs Doxorubicin Doxorubicin N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer was used for formulation of niosomes. The results indicate an unusually high stability for niosomal formulation which suggests that the active drug will only be available in vivo following niosomal degradation . Paclitaxel The oral delivery of paclitaxel niosomes prepared from different types of non-ionic surfactant showed not only increase in the stability of paclitaxel against the gastrointestinal enzymes but also high loading capacity of the niosomes system containing span 40, cholesterol and dicetylphosphate (0.0475:0.0475:0.005). In addition the formula exhibit a sort of sustained release following Higuchi model . Methotrexate Azmin et al., quoted in their research article that niosomal formulation of methotrexate exhibits higher AUC as compared to methotrexate solution, administered either intravenously or orally. Tumoricidal activity of niosomally -formulated methotrexate is higher as compared to plain drug solution. Delivery of peptide drugs Insulin Insulin entrapped niosomes composed of Brij92 and cholesterol in molar ratio 7:3 respectively showed high degree of stability against proteolytic activity of a-chymotrypsin, trypsin and pepsin in vitro. These results indicate that niosomes could be developed as sustained release oral dosage forms for delivery of peptides and proteins . Oligoneculeotide Polyethylene glycol (PEG) modified cationic niosomes were used to improve the stability and cellular delivery of oligonucleotides (OND). PEGylated cationic niosomes, composed of DC-Chol, PEG2000-DSPE and the non-ionic surfactant-Span_, offer some advantages as gene carriers. Complexes of PEGylated cationic niosomes and OND showed a neutral zeta potential with particle size about 300 nm. PEG-modification significantly decreased the binding of serum protein and prevented particle aggregation in serum. The loaded nuclear acid drug exhibited increased resistance to serum nuclease. Compared with cationic niosomes, the PEGylated niosomes showed a higher efficiency of OND cellular uptake in serum. Therefore, in terms of their stable physiochemical properties in storage and physiological environment, as well as low-cost and widely available materials, PEGylated cationic niosomes are promising drug delivery systems for improved OND potency in vivo . Ophthalmic drug delivery Gentamicin sulphate Studies showed a substantial change in the release rate and an alteration in the %EE of gentamicin sulphate from niosomal formulations upon varying type of surfactant, cholesterol content and presence or absence of DCP. In-vitro drug release studies confirmed that niosomal formulations have exhibited a high retention of gentamicin sulphate inside the vesicles such that their in vitro release was slower compared to the drug solution. A preparation with 1:1:0.1 molar ratio of Tween 60, cholesterol and DCP gave the most advantageous entrapment (92.02%+-1.43) and release results (Q8h=66.29%+-1.33) as compared to other compositions. Ocular irritancy test performed on albino rabbits, showed no sign of irritation for all tested niosomal formulations . Anti-inflammatory agents Diclofenac diethylammonium A novel elastic bilayer vesicle was entrapped with the non-steroidal anti-inflammatory drug (NSAID), diclofenac diethylammonium (DCFD) for topical use . Eighteen bilayer vesicular formulations composing of DPPC or Tween 61 or Span 60 mixed with cholesterol (at 1:1, 3:7 and 1:1 molar ratios, respectively) and ethanol at 0-25% (v/v), by chloroform filmmethod with sonicationwere developed. The elastic Tween 61 niosomes which gave no sedimentation, no layer separation, unchanged particle sizes (about 200 nm) were selected to entrap DCFD. The entrapment efficiency of the drug in the conventional and elastic Tween 61 niosomes was 65 and 93%, respectively. At least 87% of DCFD determined by HPLC remained in elastic Tween 61 niosomes when kept at 4, 27 and 45 *C for 3 months. The deformability index values of the elastic niosomeswere 13.76 and 3.44 times higher than the conventional niosomes entrapped and not entrapped with the drug, respectively, indicating the higher flexibility of the elastic vesicle especially, when entrapped with the drug. Transdermal absorption through excised rat skin was performed by vertical Franz diffusion cell at 32+-2 *C for 6 h. Gel containing elastic niosomes exhibited fluxes of DCFD in the stratum corneum (SC), deeper skin layer (viable epidermis and dermis, VED) and receiver chamber at 191.27+-9.52, 16.97+-2.77 and 3.76+-0.54_g/(cm2 h), whereas the commercial emulgel, containing an equivalent DCFD, gave 60.84+-13.63, 7.33+-1.70 and 0.14+-0.01_g/(cm2 h), respectively. The in vivo anti-inflammatory activity was evaluated by ethyl phenylpropiolate (EPP)-induced rat ear edema (n = 3). DCFD entrapped in the developed elastic niosomes and incorporated in gel gave the same ear edema inhibition percentages of 23.81% at 30 min, but 2 and 9 times more inhibition percentages at 45 and 60 min than the commercial emulgel, respectively. This result has not only demonstrated the enhancement of transdermal absorption through rat skin, but also the in vivo anti-inflammatory effect of DCFD when entrapped in the developed novel elastic Tween 61 niosomes, as well. Anti-infective agents Sodium stibogluconate Sodium stibogluconate is a drug of choice for treatment of visceral leshmaniasis which is a protozoan infection of reticuloendothelial system. Niosomal formulation of sodium stibogluconate exhibits higher level antimony as compared to free drug solution in liver. Rifampicin Niosomal formulation of rifampicin exhibits better anti-tubercular activity as compared to plain drug . Transdermal drug delivery L'OREAL the French cosmetic company considered to be the innovator of using the niosomes as delivery system for the skin when they developed a process of incorporating water or aqueous solution into a liquid lamellar phase. This process was patented by L'OREAL in 1975.  Conclusion Niosomes appeared to be a well preferred drug delivery system over liposome as niosomes being stable and economic. Niosomes have great potentiality for targeted drug delivery of anticancer, anti-infective as well as other possible delivery targets. New techniques like preparation of niosomes from proniosomes and conjugation with cell penetrating peptides should be considered for future studies. References 1. Biswal, S., et al., Vesicles of Non-ionic Surfactants (Niosomes) and Drug Delivery Potential. 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