Niosomes

Detail description about Niosomes, aquasomes, phytosomes and their preparation and Apploication tion for M. Pharm. Pharmaceutics students as per PCI syllabus

1. Niosomes, Aquasomes , Phytosomes, Electrosomes Preparation & Application Dr. Atish S. Mundada Associate Professor, SNJB’s SSDJ College of Pharmacy, Chandwad, Dist. Nashik

2. Introduction: Niosome are non-ionic surfactant vesicles obtained on hydration of synthetic nonionic surfactants, with or without incorporation of cholesterol or their lipids. They are vesicular systems similar to liposomes that can be used as carriers of amphiphilic and lipophilic drugs. Noisome are promising vehicle for drug delivery and being non-ionic are non toxic and biodegradable, biocompatible non-immunogenic and exhibit flexibility in their structural characterization. In noisome, the vesicles forming amphiphile is a non-ionic surfactant such as Span-60 which is usually stabilized by addition of cholesterol and small amount of anionic surfactant such as dicetyl phosphate. The first report of non-ionic surfactant vesicles came from the cosmetic applications devised by L’Oreal.

3. COMPOSITIONS OF NIOSOMES: The two major components used for the preparation of niosomes 1. Cholesterol & 2. Nonionic surfactants 1. Cholesterol: Cholesterol is used to provide rigidity & proper shape, conformation to the niosomes preparations. 2. Nonionic surfactants The role surfactants play a major role in the formation of niosomes . The following non-ionic surfactants are generally used for the preparation of niosomes . E.g. Spans (span 60, 40, 20, 85, 80) Tweens ( tween 20, 40, 60, 80) and Brijs (brij 30, 35, 52, 58, 72, 76). The non ionic surfactants possess a hydrophilic head and a hydrophobic tail.

4. Vesicular Drug Delivery Carriers : Novel vesicular drug delivery carriers intend to deliver the drug at a rate directed by need of body during the period of treatment, and channel the active moiety to the site of action providing targeted and controlled release of drug. Biologic origin of these vesicles was first reported in 1965 by Bingham and has been given the name Bingham bodies. The vesicles are classified on the basis of their composition : Lipoidal biocarriers 2. Non- lipoidal biocarriers

5. Comparison of Niosomes Vs Liposomes: a) Niosomes are now widely studied as an alternative to liposomes , as liposomes exhibit certain disadvantages–they are expensive, their ingredients like phospholipids are chemically unstable because of their predisposition to oxidative degradation, they require special storage and handling and purity of natural phospholipids are variable. b) Differences in characteristics exist between liposome’s and niosomes , especially since niosomes are prepared from uncharged single-chain surfactant and cholesterol whereas liposome’s are prepared from double-chain phospholipids (neutral or charged). c) Niosomes behave in-vivo like liposome’s, prolonging the circulation of entrapped drug and altering its organ distribution and metabolic stability. d) As with liposome’s, the properties of niosomes depends both on the composition of the bilayer and on method of their production. As the concentration of cholesterol increases, entrapment efficiency decreases.

6. Formation of Niosomes from Proniosomes :

7. The niosomes can be prepared from the proniosomes by adding the aqueous phase with the drug to the proniosomes with brief agitation at a temperature greater than the mean transition phase temperature of the surfactant. T > Tm Where, T = Temperature, Tm = mean phase transition temperature. Blazek-Walsh A.I. et al has reported the formulation of niosomes from maltodextrin based Proniosomes . This provides rapid reconstitution of niosomes with minimal residual carrier. Slurry of maltodextrin and surfactant was dried to form a free flowing powder, which could be rehydrated by addition of warm water.

8. Niosome Preparation Techniques: Till date various niosome preparation techniques have been reported and it has been found that niosomal properties can be affected by the preparation techniques. In general, niosomes are prepared upon hydration of a mixture of the surfactant and lipid at an elevated temperature by a hydration medium, followed by optional size reduction process and ultimately obtain a colloidal dispersion. All the common laboratory methods of niosome preparation can be classified into two major strategies. In the first strategy , volatile organic solvent is used to dissolve all the components. Removal of the organic solvent forms a thin film, which is then hydrated by an aqueous medium to obtain niosomes . The second strategy involves direct mixing of lipids and hydration medium at an elevated temperature without using any organic solvents.

9. Ether injection method: This method provides a means of making niosomes by slowly introducing a solution of surfactant dissolved in diethyl ether into warm water maintained at 60°C. The surfactant mixture in ether is injected through 14-gauge needle into an aqueous solution of material. Vaporization of ether leads to formation of single layered vesicles. Depending upon the conditions used the diameter of the vesicle range from 50 to 1000 nm. Preparation steps- Surfactant is dissolved in diethyl ether ↓ Then injected in warm water maintained at 60 o C through a 14 gauze needle ↓ Ether is vaporized to form single layered niosomes .

10. Thin Layer Hydration: (Hand shaking method) The mixture of vesicles forming ingredients like surfactant and cholesterol are dissolved in a volatile organic solvent (diethyl either, chloroform or methanol) in a round bottom flask. The organic solvent is removed at room temperature (20°C) using rotary evaporator leaving a thin layer of solid mixture deposited on the wall of the flask. The dried surfactant film can be rehydrated with aqueous phase at 0-60°C with gentle agitation. Using this method, Guinedi et al. prepared multilamellar vesicles encapsulating acetazolamide with an entrapment efficiency of about 32%; while Pardakhty et al. prepared a niosomal formulation for encapsulation of insulin. This process forms typical multilamellar niosomes .

11. Sonication Method: A typical method of production of the vesicles is by sonication of solution as described by Cable. In this method an aliquot of drug solution in buffer is added to the surfactant/cholesterol mixture in a 10-ml glass vial. The mixture is probe sonicated at 60°C for 3 minutes using a sonicator with a titanium probe to yield niosomes . Preparation steps- Drug in buffer + surfactant/cholesterol in 10 ml ↓ Above mixture is sonicated for 3 mints at 60°C using titanium probe yielding niosomes .

12. Micro fluidization Method: Micro fluidization is a recent technique used to prepare unilamellar vesicles of defined size distribution. This method is based on submerged jet principle in which two fluidized streams interact at ultra high velocities, in precisely defined micro channels within the interaction chamber. The impingement of thin liquid sheet along a common front is arranged such that the energy supplied to the system remains within the area of niosomes formation. The result is a greater uniformity, smaller size and better reproducibility of niosomes formed. Preparation steps- Two ultra high speed jets inside interaction chamber ↓ Impingement of thin layer of Liquid in micro channels ↓ Formation of uniform Niosomes.

13. 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 noisome size.

14. Reverse Phase Evaporation Technique (REV): Cholesterol and surfactant (1:1) are dissolved in a mixture of ether and chloroform. An aqueous phase containing drug is added to this and the resulting two phases are sonicated at 4-5°C. The clear gel formed is further sonicated after the addition of a small amount of phosphate buffered saline (PBS). The organic phase is removed at 40°C under low pressure. The resulting viscous noisome suspension is diluted with PBS and heated on a water bath at 60°C for 10 min to yield niosomes . Raja Naresh et al. have reported the preparation of Diclofenac Sodium niosomes using Tween 85 by this method.

15. Trans membranes PH gradient ( inside acidic ) Drug Upake Process : (Remote Loading Technique): Surfactant and cholesterol are dissolved in chloroform. The solvent is then evaporated under reduced pressure to get a thin film on the wall of the round bottom flask. The film is hydrated with 300mM citric acid (PH 4.00) by vertex mixing. The multilamellar vesicles are frozen and shared 3 times and later sonicated. To this niosomal suspension aqueous solution containing 10 mg ml of drug is added and vortexes. The pH of the sample is then raised to 7.0-7.2 with 1M disodium phosphate. This mixture is later heated at 60°c for 10 minutes so give niosomes .

16. The Bubble Method: It is novel technique for the one step preparation of liposomes and niosomes without the use of organic solvents. The bubbling unit consists of round-bottomed flask with three necks positioned in water bath to control the temperature. Water-cooled reflux and thermometer is positioned in the first and second neck and nitrogen supply through the third neck. Cholesterol and surfactant are dispersed together in this buffer (PH 7.4) at 70°C, the dispersion mixed for 15 seconds with high shear homogenizer and immediately afterwards “bubbled” at 70°C using nitrogen gas.

17. Separation of Unentrapped Drug: The removal of unentrapped solute from the vesicles can be accomplished by various techniques, which include: - 1. Dialysis The aqueous niosomal dispersion is dialyzed in dialysis tubing against suitable dissolution medium at room temperature. The samples are withdrawn from the medium at suitable time intervals, centrifuged and analyzed for drug content using suitable method (U.V. spectroscopy, HPLC etc). 2. Gel Filtration The unentrapped drug is removed by gel filtration of niosomal dispersion through a Sephadex-G-50 column and eluted with suitable mobile phase and analyzed with suitable analytical techniques. 3. CENTRIFUGATION: The proniosome derived niosomal suspension is centrifuged and the supernatant is separated. The pellet is washed and then resuspended to obtain a niosomal suspension free from unentrapped drug.

18. Factors affecting niosomes formulation: 1. 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.

19. 2. Amount and type of surfactant- The mean size of niosomes increases proportionally with increase in the HLB surfactants like Span 85 (HLB 1.8) to Span 20 (HLB 8.6) because the surface free energy decreases with an increase in hydrophobicity of surfactant. The bilayers of the vesicles are either in the so-called liquid state or in gel state, depending on the temperature, the type of lipid or surfactant and the presence of other components such as cholesterol. In the gel state, alkyl chains are present in a well-ordered structure, and in the liquid state, the structure of the bilayers is more disordered. The surfactants and lipids are characterized by the gel-liquid phase transition temperature (TC). Phase transition temperature (TC) of surfactant also effects entrapment efficiency i.e. Span 60 having higher TC, provides better entrapment.

20. 3. Cholesterol content and charge- Inclusion of cholesterol in niosomes increased its hydrodynamic diameter and entrapment efficiency. In general, the action of cholesterol is two folds; on one hand, cholesterol increases the chain order of liquid-state bilayers and on the other, cholesterol decreases the chain order of gel state bilayers . At a high cholesterol concentration, the gel state is transformed to a liquid-ordered phase. An increase in cholesterol content of the bilayers resulted in a decrease in the release rate of encapsulated material and therefore an increase of the rigidity of the bilayers obtained. Presence of charge tends to increase the interlamellar distance between successive bilayers in multilamellar vesicle structure and leads to greater overall entrapped volume.

21. 4. Resistance to osmotic stress- Addition of a hypertonic salt solution to a suspension of niosomes brings about reduction in diameter. In hypotonic salt solution, there is initial slow release with slight swelling of vesicles probably due to inhibition of eluting fluid from vesicles, followed by faster release, which may be due to mechanical loosening of vesicles structure under osmotic stress. 5. Membranes Composition- The stable niosomes can be prepared with addition of different additives along with surfactants 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.

22. In case of polyhedral niosomes formed from C16G2, the shape of these polyhedral noisome remains unaffected by adding low amount of solulan C24 (cholesterol poly-24- oxyethylene ether), which prevents aggregation due to development of stearic unhydrance . In contrast spherical Niosomes are formed by C16G2: cholesterol: solution (49:49:2). The mean size of niosomes is influenced by membrane composition such as Polyhedral niosomes formed by C16G2: solution C24 in ration (91:9) having bigger size (8.0 ― 0.03 mm) than spherical/tubular niosomes formed by C16G2: cholesterol: solution C24 in ratio (49:49:2) (6.6} 0.2 mm). Addition of cholesterol molecule to niosomal system provides rigidity to the membrane and reduces the leakage of drug from noisome.

23. Characterization of Niosomes:- a. Measurement of Angle of repose b. Scanning electron microscopy for size and morphology c. Entrapment efficiency d. Osmotic shock/ membrane rigidity e. Stability studies f. Zeta potential analysis g. In vitro drug release can be done by Dialysis tubing Reverse dialysis Franz diffusion cell

24. Applications of Niosomes: Niosomes can be used in a wide range of pharmaceutical applications due to their inherent advantages.

25. Niosomes as Drug Carriers: A number of workers have reported the preparation, characterization and use of niosomes as drug carriers. The niosomal encapsulation of Methotrexate and Doxorubicin increases drug delivery to the tumor and tumoricidal activity of the drug. In ophthalmic drug delivery bioadhesive-coated niosomal formulation of acetazolamide exhibits more tendency for reduction of intraocular pressure as compared to marketed. The chitosan coated niosomal formulation timolol maleate (0.25%) exhibits more effect for reduction intraocular pressure as compared to a marketed formulation. An increase in the penetration rate has been achieved by transdermal delivery of drug incorporated in niosomes . Niosomal entrapment increased the half-life of the drug, prolonged its circulation and altered its metabolism.

26. Immuno-Niosomes:- Niosomes can be conjugated to antibodies on their surface to form immune- niosomes . Conjugation of the monoclonal IgG antibodies to the vesicle surfaces was carried out through incorporation of a cyanuric chloride derivatized Tween 61 in the niosome formulation formed using thin film hydration techniques followed by sonication. Conjugation of the monoclonal antibody to the specific cell receptors (CD44) was demonstrated using cultured fixed synovial lining cells expressing CD44 and showed the capability of immune- niosome binding to target antigens which might provide an effective method for targeted drug delivery.

27. Magnetic Niosomes:- Niosomes show potential in combination of drug delivery and magnetic targeting in various applications particularly in cancer therapy. The basic concept of using magnetic materials in cancer therapy is to direct drug-loaded magneto- niosomes to specific organ or tissue in the body by applying extracorporeal magnets. Doxorubicin-loaded magneto- niosomal formulations were developed by encapsulating both anti- tumoral drug and magnetic material (EMG 707 ferrofluid ) into the niosome aqueous core.

28. Gene Delivery:- Although niosomes have been used in pharmaceutics since the 1980s, to date a few studies have focused on the application of niosomes for gene delivery. Niosomes have been used as cutaneous gene delivery system especially for the treatment of a variety of skin diseases. Literature reports an effective delivery of antisense oligonucleotides (OND) via cationic niosomes of Spans in a COS-7 cell line with positive results on cellular uptake of OND. DNA encoding hepatitis B surface antigen ( HBsAg ) was encapsulated into niosomes of Span 85 and cholesterol. The immune stimulating activity of these niosomal formulations were studied in terms of the serum anti- HBsAg titre and also cytokine levels (IL-2 and IFN-c) were registered following the topical application of niosomes to mice. The results revealed that niosomes can be used as DNA vaccine carriers for topical immunization which is simple, economical, stable & painless.

29. Anticancer Drug Delivery: Niosomes showed great potential in the targeted delivery of some anti-cancer drugs. Niosomes composed of a non-ionic surfactant, cholesterol and dicetyl phosphate encapsulating methotrexate (MTX) showed improvement in absorption of the drug from the gastrointestinal tract following oral administration. Improved anticancer activity or reduced toxicity of niosomal formulations of other anti-cancer agents such as vincristine , bleomycin , and paclitaxel showed that niosomes can be used as efficient drug carriers for anticancer drugs.

30. Targeting of bioactive agents: a) To reticulo -endothelial system (RES)- The cells of RES preferentially take up the vesicles. The uptake of niosomes by the cells is also by circulating serum factors known as opsonins , which mark them for clearance. Such localized drug accumulation has, however, been exploited in treatment of animal tumors known to metastasize to the liver and spleen and in parasitic infestation of liver. b) To organs other than RES It has been suggested that carrier system can be directed to specific sites in the body by use of antibodies. Immunoglobulins seem to bind quite readily to the lipid surface, thus offering a convenient means for targeting of drug carrier. Many cells possess the intrinsic ability to recognize and bind particular carbohydrate determinants and this can be exploited to direct carriers system to particular cells.

31. Delivery of peptide drugs:- Niosomal formulations used to deliver peptide drugs such as insulin and OND. Entrapment of insulin into niosomes protected it against proteolytic activity of á- chymotrypsin , trypsin and pepsin in vivo. The release of insulin was prolonged via niosomal formula prepared from Brij 92 and cholesterol. Polyethylene glycol (PEG) modified cationic niosomes which were prepared by adding PEG2000-DSPE to the cationic niosomal dispersion showed a high efficacy in cellular uptake of OND in serum. Yoshida et al investigated oral delivery of 9-desglycinamide, 8-arginine vasopressin entrapped in niosomes in an in-vitro intestinal loop model and reported that stability of peptide increased significantly.

32. Niosome formulation as a brain targeted delivery system for the vasoactive intestinal peptide (VIP):- Radiolabelled VIP-loaded glucose bearing niosomes were injected intravenously to mice. Encapsulated VIP within glucose bearing niosomes exhibits higher VIP brain uptake as compared to control. Niosomes as carriers for Hemoglobin- Niosomes can be used as a carrier for hemoglobin. Niosomal suspension shows a visible spectrum superimposable onto that of free hemoglobin. Vesicles are permeable to oxygen and hemoglobin dissociation curve can be modified similarly to non-encapsulated hemoglobin.

33. Emerging SOMES & their applications:

34. Aquasomes Aquasomes are spherical in shape with 60–300 nm particles size. These are nanoparticulate carrier systems but instead of being simple nanoparticles these are three layered self assembled structures, comprised of a solid phase nanocrystalline core coated with oligomeric film to which biochemically active molecules are adsorbed with or without modification. Aquasomes are like “bodies of water" and their water like properties protect and preserve fragile biological molecules and this property of maintaining conformational integrity as well as high degree of surface exposure is exploited in targeting of bioactive molecules like peptide and protein hormones, enzymes, antigens and genes to specific sites. These structures are self assembled by non covalent and ionic bonds. The solid core provides the structural stability, while the carbohydrate coating protects against dehydration and stabilizes the biochemically active molecules.

35. Composition of aquasomes : I- Core material Ceramic and polymers are most widely used core materials. Polymers such as albumin, gelatin or acrylate are used. II- Coating material Coating materials commonly used are cellobiose , pyridoxal 5 phosphate, sucrose, trehalose , chitosan, citrate etc. Carbohydrate plays important role as a natural stabilizer, its stabilization efficiency has been reported. III- Bioactive They have the property of interacting with film via non covalent and ionic interactions Aquasomes due to their size and structural stability, avoid clearance by reticuloendothelial system or degradation by other environmental challenges.

36. Principle of self assembly: Self assembly implies that the constituent parts of some final product assume spontaneously prescribed structural orientations in two or three dimensional space. The self assembly of macromolecules in the aqueous environment, either for the purpose of creating smart nanostructure materials or in the course of naturally occurring biochemistry, is governed basically by three physicochemical processes: the interactions of charged groups, dehydration effects and structural stability. I- Interaction between charged groups The interaction of charged groups, such as amino, carboxyl, sulphate , phosphate groups facilitates long range approach of self assembly sub units. Charged group also plays a role in stabilizing tertiary structures of folded proteins.

37. II- Hydrogen bonding and dehydration effect: Hydrogen bond helps in base pair matching and stabilization of secondary protein structure such as alpha helices and beta sheets. Molecules forming hydrogen bonds are hydrophilic and this confers a significant degree of organization to surrounding water molecules. In case of hydrophobic molecules, which are incapable of forming hydrogen bond. However, their tendency to repel water helps to organize the moiety to surrounding environment. The organized water decreases the overall level of disorder/ entropy of the surrounding medium. Since, organized water is thermodynamically unfavorable, the molecule loose water/dehydrate and get self assembled

38. III- Structural stability: Molecules that carry less charge than formally charged groups exhibit a dipole moment. The forces associated with dipoles are known as van der waals forces. Structural stability of protein in biological environment determined by interaction between charged group and hydrogen bonds. The van der waals forces are largely responsible for hardness or softness of molecules. The van der waals interaction among hydrophobic side chain promotes stability of compact helical structures which are thermodynamically unfavorable for expanded random coils. It is the maintenance of internal secondary structures, such as helices which provides sufficient softness, and allows maintenance of conformation during self assembly, small changes are necessary for successful antigen- antibody interactions.

39. Method of preparation of aquasomes : The method of preparation of aquasomes involves three steps. The general procedure consists of Formation of an inorganic core, which will be coated with carbohydrate forming the polyhydroxylated core and finally loading of the drug of choice to this assembly. I- Formation of an inorganic core II- Coating of the core with polyhydroxy oligomer III- Loading of the drug of choice to this assembly

40. I- Formation of an inorganic core:- It involves the fabrication of a ceramic core and the procedure depends upon the materials selected. For the core, ceramic materials are widely used because ceramics are structurally the most regular materials known. Being crystalline, the high degree of order in ceramics ensures that any surface modification will have only a limited effect on the nature of the atoms below the surface layer and thus the bulk properties of the ceramic will be preserved. The high degree of order also ensures that the surfaces will exhibit high level of surface energy that will favor the binding of polyhydroxy oligomeric surface film. The two most commonly used ceramic cores are calcium phosphate and diamond.

41. Preparation of ceramic core using coprecipitation : In this method, diammonium hydrogen phosphate solution is added drop wise to calcium nitrate solution continuously. The temperature of the solution is maintained at 75°C in a flask bearing a charge funnel, a thermometer and a reflux condenser fitted with a carbondioxide trap. During the synthesis, the pH of calcium nitrate has to maintain between eight and ten using concentrated aqueous ammonia solution. The precipitates are then filtered, washed and finally dried overnight. The powder was then sintered by heating to 800–900°C in an electric furnace.

42. Preparation of ceramic core using sonication: This method is based on the modification of procedure reported by Kossovsky . The solutions of disodium hydrogen phosphate and calcium chloride are mixed and sonicated using an ultrasonic bath. The ceramic core can be separated by centrifugation. After the decandation of supernatant, the core is washed, re-suspended in distilled water and filtered. The core material retained on the filter medium is collected, dried and then % yield is calculated

43. II- Coating of the core with polyhydroxy oligomer :- In the second step, ceramic cores are coated with carbohydrate ( polyhydroxyl oligomer ). The coating is carried out by addition of carbohydrate into an aqueous dispersion of the cores under sonication. These are then subjected to lyophilization to promote an irreversible adsorption of carbohydrate onto the ceramic surface. The unadsorbed carbohydrate is removed by centrifugation. The commonly used coating materials are cellobiose , citrate, pyridoxal-5- phosphate, trehalose and sucrose.

44. III- Loading of the drug of choice to this assembly:- The final stage involves the loading of drug to the coated particles by adsorption. For that, a solution of known concentration of drug is prepared in suitable pH buffer, and coated particles are dispersed into it. The dispersion is then either kept overnight at low temperature for drug loading or lyophilized after some time so as to obtain the drug-loaded formulation (i.e., aquasomes ). The preparation thus obtained is then characterized using various techniques.

45. Characterization of aquasomes : Aquasomes are characterized chiefly for their structural and morphological properties, particle size distribution, and drug loading capacity. Size distribution:- For morphological characterization and size distribution analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are generally used. Core, coated core, as well as drug-loaded aquasomes are analyzed by these techniques. Mean particle size and zeta potential of the particles can also be determined by using photon correlation spectroscopy. Structural analysis:- FT-IR spectroscopy can be used for structural analysis. Using the potassium bromide sample disk method, the core as well as the coated core can be analyzed by recording their IR spectra

46. Crystallinity :- The prepared ceramic core can be analyzed for its crystalline or amorphous behavior using X-ray diffraction. Drug payload:- The drug loading can be determined by incubating the basic aquasome formulation (i.e., without drug) in a known concentration of the drug solution for 24 hours at 4°C. The supernatant is then separated by high-speed centrifugation for 1 hour at low temperature in a refrigerated centrifuge. The drug remaining in the supernatant liquid after loading can be estimated by any suitable method of analysis. In vitro drug release studies:- The in vitro release kinetics of the loaded drug is determined to study the release pattern of drug from the aquasomes by incubating a known quantity of drug-loaded aquasomes in a buffer of suitable pH at 37°C with continuous stirring.

47. Applications of Aquasomes in drug delivery: 1. Insulin and Insulinomimetics Delivery:- The parenteral delivery of insulin through aquasomes was reported by Cherian et al. (2000) using a calcium phosphate ceramic core. Various disaccharides like cellobiose , trehalose and Pyridoxal-5-phosphate were used to coat the ceramic core. The adsorption method was used to load the drug. All formulations except cellobiose -coated particles showed prolonged reduction in blood glucose level. The highest reduction in blood glucose level was observed with Pyridoxal-5-phosphate-coated particles. Aquasomes coated with trehalose or cellobiose was reported to exhibit intermediate effect. The prolonged action was attributed to the structural integrity of peptide and slow release of drug from carrier.

48. Umashhankar et al. (2010) formulated aquasomes loaded with antidiabetic polypeptide k isolated from Momordica charantia seeds which is a well-known insulinomimetic with a remarkable anti- hyperglycaemic potential. Aquasomes were prepared by colloidal precipitation and sonication of disodium hydrogen phosphate solution and calcium chloride solution at low temperature. Thereafter, the core was primarily coated with polyhydroxy oligomers ( cellobiose and trehalose ) and subsequently loaded with polypeptide-k. An initial faster release rate was observed followed by slower release rate in second phase which was attributed to possible surface desorption of polypeptide followed by sustained release rate of polypeptide from the aquasomes matrix. It was observed that trehalose coated aquasomes released polypeptide-k faster than cellobiose coated aquasomes .

49. 2. Delivery of Antigens:- Aquasomes have also been reported for the delivery of antigens. The adjuvants generally used to enhance the immunity against antigens have a tendency either to alter the conformation of the antigen through surface adsorption or, to shield the functional groups. Thus, Kossovsky et al. (1995) formulated and tested the efficacy of an organically modified ceramic antigen delivery vehicle. These particles consisted of diamond substrate coated with a glassy carbohydrate ( Cellobiose ) film and an immunologically active surface molecule in an aqueous dispersion. These aquasomes having a size of 5-300 nm provided conformational stabilization as well as high degree of surface exposure to protein antigen. The colloidal surface provided by diamond was capable of hydrogen bonding to the proteinaceous antigen. Thus, with the help of these aquasomes , a strong and specific immune response could be elicited by enhancing the availability and in vivo activity.

50. In another report, bovine serum albumin (BSA) loaded aquasomes were prepared by self-assembling of hydroxyapatite using the co-precipitation method. The core was coated with cellobiose and trehalose , and finally BSA was adsorbed as model antigen onto the coated core. The aquasomes were found to be spherical in shape with diameter around 200 nm. About 20-30 % drug was loaded in the prepared aquasomes . The immunopotentiating activity revealed that the aquasome formulation had a higher activity as compared to plain BSA. Aquasomes of malarial merozoite surface protein 119 (MSP-119) were prepared by adsorption of antigen on self-assembling hydroxyapatite carriers. The use of ceramic core based nanodecoy system as an adjuvant and delivery vehicle for hepatitis B vaccine for effective immunization was reported by Vyas et al. (2006).

51. 3. Delivery of Enzymes:- A nanosized ceramic core-based system for oral administration of the acidlabile enzyme serratiopeptidase was prepared by colloidal precipitation under sonication at room temperature ( Rawat et al. 2008). The core was then coated with chitosan under constant stirring, after which the enzyme was adsorbed over it. The enzyme was protected by further encapsulating the enzyme-loaded core into alginate gel. The TEM images of particles revealed that they were spherical in shape, with an average diameter of 925 nm. The enzyme-loading efficiency of the particles was found to be approximately 46%. The aquasomes were found to be protecting the structural integrity of enzymes so as to obtain a better therapeutic effect.

52. 4. Delivery of Non-Protein Molecules Encouraging results with protein/peptide drugs have led to the use of aquasomes for the conventional drugs including anti-cancer drugs ( Hiremath et al. 2011). Nanjwade et al. (2013) showed that etoposide is preferentially targeted to liver, spleen, lungs and kidney when administered as aquasomes . topical cream formulation of aquasomal dithranol has been reported to lead to a more sustained release of the drug from its formulation ( Tiwari et al. 2012).

53. Phytosomes The term phyto means plant while some means cell-like. Phytosomes are also known as herbosomes . Phytosome is composed of phospholipids, mainly phosphatidylcholine, producing a lipid compatible molecular complex with other constituents. Phytosomal complexes were first investigated for cosmetic applications. But PHYTOSOME process was developed and petended by Indena , a leading supplier of nutraceutical ingredients like milk thistle, ginkgo biloba , grape seed, green tea, hawthorn, ginseng etc. Phytosomes‖ are better absorbed and utilized, as a result produce better results than conventional herbal extracts. Presently phytosomes are used primarily in cosmetics to deliver water soluble substances to the skin.

54. Phytosomes are not liposomes; structurally, the two are distinctly different. The phytosome is a unit of several molecules bonded together, while the liposome is an aggregate of many phospholipid molecules that can enclose active phytomolecules . But without specifically bonding to them. This technology is also useful in pharmaceutical formulations intended for treatment of oral cavity in which the contact times are very short because phospholipid allows a greater adhesion of the product itself to the surfaces it comes into contact with. Phytosomes are advanced herbal products produced by binding individual component of herbal extract to phosphatidylcholine resulting in a product that is better absorbed and produces better results than the conventional herbal extracts. The effectiveness of any herbal product is dependent upon delivering an effective level of the active compounds. Phytosome has an added dimension; the proven health giving activity of the phospholipids themselves.

55. The phytosome process has been applied to many popular herbal extracts including Ginkgo biloba , grape seed, hawthorn, olive fruits and leaves, milk thistle, green tea, ginseng, kushenin , marsupsin and curcumin . The poor absorption of flavonoid nutrients is likely due to two factors. First, they are multiplexing molecules too large to be absorbed by simple diffusion, while they are not absorbed actively. Second, flavonoid molecules typically have poor miscibility with oils and other lipids, severely limiting their ability to pass across the lipid-rich outer membranes of the enterocytes . Water-soluble flavonoid molecules can be converted into lipid-compatible molecular complexes, aptly called phytosomes . Phytosomes are better able to transition from a hydrophilic environment into the lipid-friendly environment of the enterocyte cell membrane and from there into the cell. Finally reaching the blood

56. Milk Thistle: The First Phytosomes: The first commercial phytosome preparation was based on the flavonoHgnan silybin , the major constituent of silymarin . A flavonol complex extracted from the milk thistle fruit ( Siiyhum marianum , family Asteraceae / Compositae ). This phytosome preparation was initially christened IDB 1016 or Silipide and subsequently recast as Siliphos * Phytosome ™. Silybinphosphatidylcholine is clinically validated for its antioxidant, anti-inflammatory and liver detoxification benefits. The phytosome technology has revolutionized the nutraceutical industry by serving the following benefits: 1. Phytosomes produces a little cell where the valuable components of herbal extracts are protected from destruction by digestive secretions and gut bacteria. 2. It assures proper delivery of drug to the respective tissues.

57. Method of Preparation: Phytosomes are prepared by reacting 3–2 moles (preferably with one mole) of a natural or synthetic phospholipid, such as phosphatidylcholine, phosphatidyl- ethanolamine or phosphatidyiserine, with one mole of phytoconstituents either alone or in the natural mixture in an aprotic solvent, such as dioxane or acetone, in a 1:2 or 1:1 ratio. The optimum ratio of phospholipid to phytoconstituent is 1:1. The complex thus formed can be isolated by precipitation with an aliphatic hydrocarbon or lyophilization or spray drying.

58. Characterization and Evaluation of Phytosome : Phytosomes are characterized for physical attributes, i.e. shape, size, its distribution, percentage drug capture, entrapped volume, percentage drug release, and chemical composition. Hence, behavior of Phytosomes, in both physical and biological systems is governed by factors such as physical size, membrane permeability; percent entrapped solutes, chemical composition, quantity and purity of the starting material. a)Visualization: Visualization of phytosomes can be achieved using transmission electron microscopy. b)Entrapment efficiency: The entrapment efficient of a drug by phytosome can be measured by the ultracentrifugation technique. c)Transition temperature: The transition temperature of the vesicular lipid systems can be determined by differential scanning colorimeter.

59. d) Surface tension activity measurement: The surface tension activity of the drug in aqueous solution can be measured by the ring method in a Du Nouy ring tensiometer . e) Vesicle stability: The stability of vesicles can be determined by assessing the size and structure of the vesicles over time. The mean size is measured by Dynamic Light Scattering (DLS) and structural changes are monitored by Transmission Electron Microscopy (TEM). f) Drug content: The amount of drug can be quantified by modified high performance liquid chromatographic method or by a suitable spectroscopic method. g) Vesicle size and Zeta potential: The particle size and zeta potential can be determine by DLS using a computerized inspection system and photon correlation spectroscopy

60. Applications of Phytosomes: 1. Enhancing Bioavailability: Phytosomes of Evodiamine , a quinoline alkaloid, ( Evodia rutaecarpa )possess a multitude of pharmacological activities, such as anti-tumor, anti-inflammatory, anti- nociceptive , anti-obesity and thermoregulatory effects proved to have higher in vitro dissolution rate, better absorption, longer action time and higher bioavailability. A prolonged action time and higher bioavailability was observed due to extended release of the drug from the phytosome . Moreover, these phytosomes might reduce the first-pass metabolism of Evodiamine by bypassing liver and therefore avoiding the direct contact of the drug with the hepatic metabolism enzymes. A European patent by ‘ Indena S.P.A.’ relates to novel phospholipids complexes of olive fruits or leaf extracts having improved bioavailability.

61. Ellagitannins belongs to a class of polyphenols which possess antioxidant mechanisms against cardiovascular disorders, cancers, and also wound healing, due to their antibacterial and anti-viral activities. The Papp (Apparent permeability) values for ellagitannins and its phytosomes by Everted gut sac technique were found to be 60 and 135 nm/s respectively. This showed that the apparent permeability of ellagitannins increased almost two times on complexation with Phosphatidylcholine. The factors responsible for poor oral bioavailability of the phytoconstituents could be the dosage form, low concentrations available after chemical degradation, physical inactivation and elimination through the gut wall and liver. However, the enhanced bioavailability of phytosomes administered through oral route is a result of increased hydrophilicity , solubility, decreased hepatic metabolism and enriched drug absorption in the systemic circulation.

62. 2. Antioxidant properties S. Moscarella et al 1993 studied the antioxidant and free radical scavenging activity of Silipide which is phytosome of Silybum marianum plant against liver oxidative damage induced by CC14 and paracetamol (high dosages) in rats. Serum Malondialdehyde levels of patients treated with silipide showed a remarkable reduction (36%) after 2 months of therapy suggesting that it possesses anti- lipoperoxidant activity against free radical attack in humans. A physically stable phytosomal formulation of Quercetin was prepared with higher encapsulation efficiency and physical stability to improve its efficacy in intestinal absorption and its preservation from oxidation in foodstuffs. A metal phytosome synthesized by encapsulation of extract of Calendula officinalis depicted that the cell viability was approximately 35% and 81% of the plant extract and Au-loaded phytosome respectively. .

63. 3. Hepato -Protective: The leaf extracts of Ginkgo biloba (Family: Ginkgoaceae ) have been found to possess cardioprotective , anti-asthmatic, anti-diabetic, anti-oxidant, hepatoprotective and potent CNS activities. The study demonstrated that phytosomes of G. biloba (200 mg/kg) significantly alleviated isoproterenol -induced myocardial necrosis. Mangiferin (MF) showed potent scavenging activity on Diphenyl-1-picrylhydrazyl (DPPH) radicals which stimulates liver regeneration in various liver injuries. Ex vivo study showed significant increased absorption of MF from MF herbosomes as compared to plain MF. Naik et al. (2008) investigated about the protective effects of Ginkgoselect Phytosome ® on Rifampicin (RMP) induced hepatotoxicity and the probable mechanism(s) involved. Hepatoprotective effect of Ginkgo select Phytosome ® in RMP induced oxidative damage may be linked to its antioxidant and free radical scavenging activity.

64. 4. Cancer treatment: The chemical components like flavones, isoflavones , flavonoids , anthocyanins , coumarins , lignins , catechins , and isocatechins of medicinal plants mainly possess antioxidant properties that contribute to their anticancer potential. S. Shalini et al. 2015 researched on methanolic extract of Terminalia arjuna bark and its phytosome to investigate its antiproliferative activity on human breast cancer cell line MCF-7 by MTT assay by comparing its activities with Quercetin and its phytosomes . The IC50 values of the extract and its phytosome were 25μg/ml and 15μg/ml respectively which suggests that they exert more antiproliferative effect as compared to free drug. Narges Mahmoodi et al. 2014 worked with Silybin and its phytosomes to study the expression levels of estrogen receptor α ( ERα ) overexpressed in breast cancer, which is responsible for tumor growth enhancement, and is a prognostic and predictive factor.

65. 5. Transdermal application: Malay K Das et al. 2104, investigated about Rutin , one of the most common flavonoid ( Ruta graveolens ) used to treat capillary fragility, uv radiation-induced cutaneous oxidative stress and IT possesses antioxidant, anti-inflammatory, antithrombotic, antineoplastic , and antiplatelet activity. It was observed that the Rutin phytosomes were better able to penetrate the impermeable stratum corneum than its free form. Skin uptake of Rutin phytosomes was 33 ± 1.33 % whereas that of Rutin was 13 ± 0.87 %. The phytosomal complex of saponins and plant extracts ( Panax ginseng M.) proved to be more active in vasal protection, capillary permeability, protection against UV radiation. Also helpful in preparation of dermatological and cosmetic pharmaceutical formulations, exhibiting moisturizing effect on the cutis making it more elastic due to a fibroblastic stimulation at the dermal level, with an increase in proteoglycan and collagen synthesis.

66. 6. Wound healing A. Mazumder et al. 2016 studied the wound healing activity of Sinigrin , one of the major glucosinolates occurring in the plant of Brassicaceae family, when evaluated as alone and as a phytosome complex on HaCaT cells. The sinigrin–phytosome complex shows complete recovery of wound (100%) whereas the phytoconstituent alone displayed only 71% healing. S. Lakshmi Devi et al. 2012, researched on the comparative effects of ethanolic extracts of Wrightia arborea leaves and its phytosomes . The phytosomes exhibited about 90.40% healing while the ethanolic extract alone could heal only 65.63% of the wound.

67. Electrosomes Biofuel cells are electrochemical devices that use enzymatic reactions to catalyze the conversion of chemical energy to electricity in a fuel cell. They can be classified as microbial fuel cells (MFCs), which use living microorganisms, or enzymatic fuel cells, which use purified enzymes. The electrosome, a novel surface-display system based on the specific interaction between the cellulosomal scaffolding protein and a cascade of redox enzymes that allows multiple electron-release by fuel oxidation. The electrosome is composed of two compartments: ( i ) a hybrid anode, which consists of dockerin -containing enzymes attached specifically to cohesin sites in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid cathode, which consists of a dockerin -containing oxygen-reducing enzyme attached in multiple copies to the cohesin -bearing scaffoldin .

68. In situ processing of complexed polysaccharides such as starch into fuel was demonstrated in a hybrid cell by the coupling of yeast displaying the starch-hydrolyzing enzyme glucoamylase with glucose oxidase -displaying yeast in a mixed culture. In the microbial fuel cells, the metabolic pathways of the organism provide full cascades of redox enzymes, which can catalyze the full oxidation of various fuels. The microorganisms grow and divide and that keeps the system alive and able to generate more catalyst for long-term usage. The use of enzymatic cascades in enzymatic fuel cell anodes resulted in very high power outputs, as the electron density achieved was much higher when the fuel was fully oxidized; thus all electrons extracted from a fuel molecule could be transferred to the anode. Substrate-channeling methods involve the design of systems in which several catalysts are designed to act in proximity, which can significantly improve the total efficiency of a cascade of reactions.

69. The scaffoldin protein consists of several modules named cohesins , which bind with high affinity to the complementary dockerin modules borne by the cellulosomal enzymes. The binding of the scaffoldin to the bacterial cell occurs via a second type of dockerin module in the scaffoldin protein, which binds to a cohesin module of an anchoring scaffoldin bound to the cell surface. In a defined bacterial species, different cellulose-degrading enzymes with complementary activities share similar dockerin modules of like specificities, and the enzymes are thus bound randomly to the scaffoldin protein, generating heterogeneous cellulosomes . Scaffoldin chimeras could also be generated by fusing genes encoding for cohesins from different microorganisms via short protein linkers, thus generating designer cellulosomes .

70. The gene encoding for the chimeric scaffoldin can be surface-displayed in microorganisms, thus enabling the attachment of multiple enzymes. The approach of scaffoldin surface-display enables an increase of the density of the cathodic biocatalyst on the yeast surface by virtue of a scaffoldin protein that contains several cohesin domains displayed using the YSD system, to which multiple copies of a single type of a dockerin -containing oxygen-reducing enzyme are bound. Depicted in Figure 1 is the electrosome that was designed for use both in an anode and a cathode compartment; in each compartment, the unique attributes of the cellulosome scaffoldin give a different advantage.

71. In the anode (Figure 1A), the ethanol oxidation cascade consists of two enzymes, ADH and formaldehyde dehydrogenase ( FormDH ), both containing a different dockerin module of Acetivibrio cellulolyticus and of Clostridium thermocellum ) C. thermocellum ( ( zADH -Ac and pFormDH -Ct), respectively, assembled on a ‘ designer’-scaffoldin chimera displayed on the surface of S. cerevisiae . At the cathode (Figure 1B), copper oxidase ( CueO ) was selected for surface-display. CueO is a multi-copper oxidase enzyme expressed by E. coli that catalyzes the oxidation of Cu(I) ions coupled to oxygen reduction to water. This enzyme is promiscuous and thus can oxidize different aromatic compounds, some of which can act as redox mediators in the cathode compartment of a biofuel cell.