Transethosomes and Nanoethosomes: Recent Approach on Transdermal Drug Delivery System Transethosomes and Nanoethosomes: Recent Approach on Transdermal Drug Delivery System

In the past few decades, an emerging drug delivery system that came into light is transder- mal drug delivery system. It has become the talk of the town in the field of drug delivery because of its better and easy accessibility. Though it is one of the attractive routes, trans- port of drug through the skin has remained a challenge. To overcome the challenge, vesicular system has been adopted so as to have better skin permeation of bioactive agents. Vesicular system like liposome has shown inefficiency to cross the layers of skin. Then transethosomes and nanoethosomes are employed for delivering drug into the deeper layer of skin. Nanoethosomes and transethosomes have same composition that is water, ethanol and phospholipid. Transethosome contains edge activator additionally. Due to the presence of ethanol and edge activator, it displayed enhanced skin permeation. Vesicular system gives a better patient compliance, being a non-invasive method of drug administra- tion. In this chapter, we attempted to provide brief information about methods of preparation, characterization and pharmaceutical uses of nanoethosomes and transethosomes.


Introduction
In last few decades many significant advancement in the field of drug delivery technology have been made. This advancement took place as there was no remarkable growth in developing new drug entities. Drug delivery emerged as a branch of science which comprises of biopharmaceutics and pharmacokinetics. Drug delivery technology is accepted as it imparts therapeutic as well as economical values to the health care products. The above said characteristic of drug delivery technology helps the old drugs to stand the competition of growing market. Drug delivery enhances the efficacy of drugs through controlled release by considering the factors like carrier system, route of administration and target of drug action. Drug delivery system improves patient compliance, therapeutic index and bioavailability [1].
In present scenario researchers are working on delivering the active pharmaceutical ingredient through transdermal route but still the effective delivery of drug through the said route is challenging. Transdermal drug delivery system originated in the year 1950 in U.S. In transdermal system drug permeate from the outer layer of skin to the blood stream through various layers of skin [2]. Transdermal drug delivery system is advantageous over traditional method. Drug administered through transdermal drug delivery system does not pass through GI Tract so are not exposed to degradation in the gut. As transdermal system delivers drug directly into blood stream through layers of skin, so drugs bypasses pre-systemic metabolism. Transdermal system is most suitable for drugs with short half life and narrow therapeutic range. The system helps in maintaining controlled level of plasma, in case of toxicity drug administration can be easily stopped. Few disadvantages are also attached to this system, such as drugs requiring blood volume cannot be administered [3][4][5][6][7]. Due the patches and the adhesive used in it, few recipients may experience skin irritation. For some section of patients it may not be economical. In the process of developing transdermal drug delivery system few basic areas has to be considered such as drug's bioactivity, the characteristics of skin, formulation employed, adhesive to be used and the design of the system. Permeation of drug through skin is dependent on factors like structure of skin and properties associated with it, the physicochemical property of the penetration molecule in relationship with the skin and lastly the system that carries the drug to the skin for penetration [8][9][10].
For overcoming the limitation of barrier function of skin some methods are undertaken such as iontophoresis, sonophoresis, electrophoresis along with micro-invasive techniques, vesicular system and employment of permeation enhancers [11,12]. Transdermal system of drug delivery transports bioactive agents directly into blood stream. Transdermal system has many benefits like it bypasses first pass metabolism, it ensures better patient compliance and with this system there is mere chances of any kind of tissue injury. Currently vesicular system is mostly studied approach for transdermal drug delivery system. Vesicular system contains vesicles which are composed of colloids with hydrophilic and amphiphilic groups. Hydrophilic part makes the core and it is guarded by amphiphilic part in a bilayer effusion. The vesicular system act as a carrier for drugs which may be hydrophilic, lipophilic or amphiphilic. The vesicles have the ability of encapsulating the said drug types. Factors such as size, lamellarity, thermodynamic phase, surface charge are determinant of the efficiency of vesicular system as carrier [13].
Presently various type of vesicular carrier systems are available such as liposomes, ethosomes, transferosomes, niosomes, transethosomes etc. (Figure 1). All of them have their particular merits and demerits. There is not a single vesicular carrier that can fulfill all the criteria of drug delivery. In this chapter we are mainly focus on transethosomes and nanoethosomes vesicular carrier system.

Transethosomes
Liposomes, which are conventional lipid-based vesicular system, are not capable of crossing the stratum corneum. As they exhibit low permeation into deeper layer of skin, eventually they get accumulated on superior layer of stratum corneum. To improve the skin permeation, liposomes are added with edge activators like Span 80, Span 25, Tween 80, sodium cholate and are named as transferosomes. Transferosomes enhances skin permeation due to their deformable ability but they are unable to penetrate deep into the stratum corneum. Another vesicular system called ethosomes is also available. Ethosomes are composed of phospholipid, ethanol and water ( Table 1). Due to presence of ethanol the intercellular space between the corneocytes increases which increase the permeation [14][15][16].
Transethosomes are the combination of transferosome and ethosomes. Transferosomes shows both the quality of becoming deformable and skin permeation. This vesicular system was introduced in the year 2012 by Song et al. Transethosomes can be taken by topical as well as  systemic route. Drugs ranging from low molecular weight to high molecular weight can easily be entrapped by this system. As the bioactive agent is protected due to encapsulation, so it releases its content in a very slow and gradual manner. Due to their biodegradable and biocompatible nature, it shows highly efficient entrapment ability. Its preparation does not require any undue pharmaceutical additives and no tedious process is involved in it [15][16][17].

Nanoethosomes
Ethosomes are non-invasive carriers that assist the bioactive agents to penetrate deep into the layers of skin or into the blood stream [18]. This is a modified version of liposomal carrier system. They contains lipid vesicle along with water and ethanol. Ethosome was developed in the year 1996 by Touitou [19]. Ethosomes are known to release the entrapped bioactive agents in a constant and gradual manner. Ethosomes comes in various size. Ethosomes of nanometer size are called nanoethosomes. Presence of high content of ethanol imparts a negative charge on surface of vesicles which promotes reduction of its size [20]. Due to smaller size, nanoethosomes easily penetrate through the intercellular space [6].

Advantages and disadvantages of vesicular carriers
Many vesicular formulations for drug administration through parenteral, topical as well as oral route have been developed. Vesicular drugs provides advantages like convenience, safer way of drug administration and most importantly it provides a protection for the active constituent in in-vivo condition from premature degradation. In addition to the said advantages vesicular carriers makes it possible to release encapsulated molecule in sustained and controlled manner. Due to this pattern of release it becomes easy to ensure targeted delivery of drug to the target tissues. Challenges like pre-systemic metabolism, frequent dosing, and variation in GI absorption of drug can be overcome by vesicular carriers. Vesicular carriers reduce the dosing frequency due to which the cost to the patient decreases and ensures better

Advantages and disadvantages of transethosomes
Advantages of transethosomes are that it provides semisolid dosage form for administration which gives a better patient compliance. Transethosomes ensures an enhanced permeation of drug through skin. It bypasses presystemic metabolism. Few disadvantages of transethosomes are like dermatitis, allergic reaction or skin irritation in some patients. Some product gets lost as it is transferred from alcoholic media to aqueous media [21,22].

Advantages and disadvantages of nanoethosomes
Nanoethosomes as a carrier makes it possible to deliver large molecules like proteins. The material used to prepare nanoethosomes is non-toxic, so it is not at all harmful for the recipient. As it is easy to use, so it provides better patient compliance. As compared to methods like phonophoresis, iontophoresis it is simple method of drug delivery. Few disadvantages are also associated with nanoethosomes such as it does not provide a rapid bolus drug input. To gain entry into blood circulation the drug has to be soluble in both lipophilic and aqueous phase. Adhesives used in nanoethosomes may not adhere on the skin of every single patient. A particular molecular size of drug can be can be delivered by this system. It may not be economical for certain segment of patients [23][24][25]. More stable, high penetration due to high deformability, biocompatible and biodegradable, suitable for both low and high molecular weight and also for lipophilic as well as hydrophilic drugs and reach up to deeper skin layers.
None, but for some limitations

Transethosomes
• Enhanced drug permeation through skin for transdermal drug delivery.
• Raw material in the formulation is non toxic in nature.
• More stable • The transethosomal drug is administrated in a semisolid form.
• Avoidance of first pass metabolism • Biocompatible and Biodegradable • Product loss during transfer from alcoholic and water media.
• Skin irritation or allergic reaction on contact dermatitis.

Challenges of vesicular carriers
Though vesicular carriers are becoming popular day by day but due to chemical reactions like oxidation and hydrolysis of the phospholipid component of vesicular carrier, its stability is a matter of concern. The physical and chemical instability can be seen by events like leakage of encapsulated drug and change in vesicle size which takes place due to fusion and aggregation.

Comparison with different vesicular carriers
The comparison of nanoethosomes and transethosome with other vesicular carriers are described in Table 2.

Ethanol effect on skin
Nanoethosomes and transethosomes contains around 20-50% of ethanol [26]. The action of ethanol on the lipid layer is shown in Figure 2 with comparison to the lipid layer where there is no ethanol present. Figure 2B is showing void space is created and filled with ethanol that in turn increased area per lipid molecule [27]. X-ray diffraction shows that the lipid bilayer has interdigitated Membrane leaflet which eventually leads to thinner membrane when ethanol comes in contact with the lipid bilayer. As the surface density of lipid decreases the bilayer gets thinner which leads to membrane distention. Due to presence of alcohol at the surface the change in membrane shape get accelerated. Chanturiya et al. experimentally proved that alcohol promote fusion of discontinuous membrane by breaking the single layer continuity. This study do not confirm about alcohol accelerating post fusion membrane distention [28]. The result that comes up from this demonstration is that presence of ethanol can bring about alteration in rate of change in shape by membrane in an exocytosis manner. In this demonstration the concentration of alcohol is quite higher as compared to that which is found in the blood in case of intoxication. Ethanol does accumulate in some region of body such as striatum, brain to an extent of three times of the level that is found in blood. Clearance from alcohol from striatum is relatively slower s compared to other region of body. This characteristic may affect chronic alcoholics during an event of binge drinking as the kinetics of neurotransmission may have an implication of the said characteristics [29]. This also suggests that the deformable nature of vesicle assist in enhanced penetration and drug delivery of drug. Godin and Touitou [30] proved vesicle adsorption to the skin and fusion of vesicular layer with stratum corneum of skin. It is observed that the drug present in liposomal preparation is unable to penetrate into skin although the preparation gets adsorbed to the membrane. In case of ethosomal preparation the ethanol present in it gets intercalated on lipid present in stratum corneum which results in increase in membrane permeability. Ethosomes are flexible and after fusion with the membrane they successfully deliver the drug inside the cells [31]. The main parameter that defines the movement of transfersomes across the cutaneous is extent of flexibility and partition coefficient. The passage of transferosomes through the stratum corneum layer is governed by hydration force. The fluid content of stratum corneum is lower than other layer of skin due to which fluid gradient created. Cevc et al. explained the passage through skin by osmotic strength theory [14]. The active ingredient can easily release in stratum corneum and diffuse through different layer of skin and reaches blood circulation. Transferosomes pass through the intercellular space of stratum corneum by deforming itself. No studies reported that transferosomes can permeate through the skin without altering its shape. The vesicular carrier transethosome makes its pathway through the stratum corneum into the deep skin layer by adhering to lipid lamella after interacting with the disturbed layer of stratum corneum. The presence of ethanol and edge activator in vesicles enhances its flexibility and fluidity and due this kind of elastic nature, vesicles can easily pass through narrow intercellular pathway. The permeation mechanism of different type of vesicular carriers is explained in Figure 3.

Method of preparation
There are four methods of preparation for nanoethosomes and transethosomes which are explain in here.

Preparation by cold method
In this method, ethanol is taken and to it phospholipid is added then by constant stirring it is dissolved at room temperature. Then to this solution propylene glycol is added and stirred vigorously. Then the solution mixture is heated up to a temperature of 30 C. Simultaneously on other hand water is being heated with up to a temperature of 30 C. Water and ethanol both solvents are used so that drug according to its affinity can dissolve in either of them. Stirring is continued to next 5 minutes. Then the suspension obtained is cooled down at maintained at room temperature. In the final step, by employing methods like extrusion or sonication, the vesicle size can be adjusted. Then the prepared ethosomal vesicles are kept in refrigerator for storage [32,33].

Preparation by hot method
In hot method, a dispersion of phospholipid and water is subjected to heating at a temperature of 40 C. The step of heating at the same temperature is continued till colloidal solution is formed. Simultaneously in another vessel two more solvent namely glycol and ethanol is added and heated at a 40 C. When both mixtures steadily attain a temperature of 40 C then to the aqueous phase the organic phase is added. Then stirring is done up to next 5 minutes and the obtained vesicle suspension is cooled down to room temperature. Depending on the ability and affinity of drug to bind with hydrophilic and hydrophobic solvent, it either gets dissolved in water or in ethanol. The required size of ethosome vesicles can be easily prepared by using methods like extrusion or sonication [6].

Preparation by classical method
Here in this method, a mixture of ethanol, active medicaments and phospholipid is taken in such way that the active medicament and phospholipid get dissolved in ethanol. Then the solution mixture is heated by using a water bath at a temperature of about 30 AE 1 C. In the next step to solution mixture double distilled water is added with continuous stirring at a speed of 700 rpm. Then with the help of hand extruder, the obtained vesicles are homogenized for three cycles using polycarbonate membrane [34].

Preparation by mechanical dispersion method
In this method a mixture of chloroform and ethanol is taken in a round bottom flask (RBF). To the round bottom flask soya phosphatidylcholine is added and made to dissolve in the chloroform and ethanol mixture. By using rotary vacuum evaporator organic solvent is removed. This step is carried out at a temperature that is above the lipid transition temperature. The main purpose of maintaining that temperature is that at the said temperature a thin lipid film gets deposited on the surface of a round bottom flask. Then the round bottom flask is kept overnight so that trace of solvent can be obtained from the lipid film that got deposited on the round bottom flask. Then hydroethanol hydration is being done by simply rotating round bottom flask at the required temperature by employing different concentration of drug mixture [35].

Morphology
Morphology defined as study of shape and size of vesicular carriers. Generally vesicular carriers are regular spherical in shape and they are physically soft and flexible and core is enclosed. On the basis of formulation, vesicular carrier may be small, unilamellar or multilamellar. With the help of microscope morphology of vesicular carrier is studied. As most of the vesicles are nano-sized so to view the morphology scanning electron microscopy is used. In addition to identification study, morphology also explains the detection the pattern of packing of particles and aggregation [36].

Particle size and size distribution
As smaller size increases the efficiency of drug delivery to the desired site, vesicles in the vesicular carriers are of nanometer to micrometer range. Uniform size is major factor to consider and it can be achieved depending on instrumentation and formulation procedures. Nanosizers are employed to measure the size and size distribution of vesicular carriers. Photon correlation spectroscopy is also used for determination of particle size [18].

Zeta potential
Distribution of charge on the surface of vesicular carrier is expressed by zeta potential. The presence of charge on the surface of nanoparticle is a major determinant of stability of the product. The presence of negative and positive charge on vesicular carrier depends upon the excipient used in the formulation. Zeta potential can be defined as the degree of electrostatic repulsion and attraction in colloidal dispersion. Zeta potential provides information regarding every components of formulation and interaction among them. It also gives information regarding surface chemistry. Zeta potential is determining factor for stability of colloidal dispersion system. It also determines the interaction between vesicles and membrane [37,38].

Drug content
To ascertain whether the preparation content the required active ingredient in required amount in the vesicles, the vesicles are lyses so that the content is released. The released content is put into the solution then the solution is subjected to spectrophotometric analysis or chromatographic assay. Lysis of vesicles is done by solvents like isopropylalcohol, methanol, etc. [39].

Loading capacity, encapsulation efficiency, and vesicle yield
To have proper information about the drug that is actually trapped in the vesicles, entrapment is determined. For determination of entrapment, from the formed vesicles unentrapped drug is separated. Separation is done either by elution method using elution technique by the help of suitable column or by simply by the method of ultracentrifugation. Centrifugation is supposed to be done in such a controlled speed and temperature so that the vesicles do not get ruptured during the process. At end of centrifugation the supernatant produced is separated from the vesicles. Then the vesicles are treated with solvent likes triton-X. 2-propanol so that it get lysed then drug content is subjected to UV vis spectroscopy, the amount of drug entrapped is calculated [40]. The encapsulation efficiency (EE), Loading capacity (LC) and Vesicle Yield are calculated by the following formula's.
Where Ao is the initial amount of the drug used, Au is the nonencapsulated drug, W is the amount of lipid material used in the vesicular formulation and Av is the amount of vesicular carrier produced [41].

Drug release and biophysical interactions
Drug release is dependent on many factors such as the additives used in formulation and also the physiological condition at the site of administration. As we can consider when the particle size is relatively larger and the vesicular carriers the non-flexible then their permeation across the biological membrane is hampered. Every single body part possess its particular pH, if the formulation that has to be administered in that area is in that pH range then physiological functioning is not affected but in case the formulation has pH different from the physiological pH of concerned area then physiological condition would get affected. pH can cause ionization of drugs that can result in inability of the drug to pass through the biological barrier [42,43].

Stability
The stability of the preparation can estimate by ensuring whether there is any change in vesicle size. When stability is concerned, it is believed that homogeneous preparation is more stable as compared to heterogeneous ones. Another way of determining the stability of preparation is to study its membrane stability and molecular arrangement by X-ray scattering or differential scanning method. Study of particle size is just considered as a quality control test not a test to ascertain the internal stability of preparation. This conclusion is made as it is seen in properly lyophilized liposome, where particle size is stable but additive like cryoprotectant interfere with lipid layer molecular arrangement in bilayer membrane [44].

Application of nanoethosomes and transethosomes
Ethosomes and nanoethosomes have enhanced efficiency as compared to liposomes. They give 65% better results in drug delivery as compared to liposomes as they easily crosses the human skin layers. Few bioactive agents are considered to investigate the efficacy of these vesicular systems which are described in Tables 3 and 4 [45].

Transdermal delivery of anti-fungal bioactives
To ascertain the drug delivery efficiency of vesicular carrier system for anti-fugal drugs, Bhalaria et al. conducted an experiment where they took fluconazole and prepared nanoethosomes from them [46].  Table 3.
Applications of nanoethosomes for transdermal delivery of various classes of drugs. 45% ethanol there is higher level of entrapment and vesicles formed are of optimized size [47]. Table 3 provides information regarding few of the researches that has been conducted on nanoethosomal drug delivery of anti-fungal class of drug.

Transdermal delivery of anti-inflammatory bioactives
To have an idea about the efficacy of ethosomal preparation in delivering anti-inflammatory drug Paolino  permeation, better tolerability of skin, and in vivo consideration showed enhanced antiinflammatory activity in human volunteers. Nanoethosomes being smaller in size and having higher content of ethanol displayed better entrapment efficiency. Brief information has been given in Table 3 regarding transdermal delivery of anti-inflammatory drug [48].

Transport of drug with action on cardiovascular system
To ascertain the extent of delivery of cardiovascular drug from transdermal system of drug delivery Touitou et al. investigated the efficiency of nanoethosomes filled with minoxidil. The nanoethosomes that were prepared with 2% of phosphatidylcholine and 30% of ethanol displayed a relatively increased permeability when compared with the formulation of hydroethanolic and phospholipid minoxidil. In another experiment Ahad et al. took valsartan and investigated the penetration capability of the nanoethosomes containing valsartan in the skin of Wistar albino rats. The results were amazing as there was increased penetration into deeper layers of skin when compared with liposomal preparation of same drug and it also shows increased bioavailability when compared with oral route of administration. In addition to this Bhosale and Avachat documented an increased antihypertensive action in Wistar albino rats when given transdermally as compared with oral administration of same [49]. Table 3 shows role of nanoethosomes in antihypertensive drug delivery.

Transport of bioactive agent with antiviral activity
Nanoethosomes of lamivudine is been prepared by Jain et al. to evaluate the efficacy of transdermal delivery of antiviral drugs. The nanoethosomal preparation showed 25 times better penetration of drug through as compared to a simple solution. T-lymphocytes showed increased uptake ability for nanoethosomes compared to free drug solution [33]. Few more examples regarding transdermal drug delivery of antiviral drug is given in Table 3.

Delivery of miscellaneous bioactives
Nanoethosomes were compared with hydroethanolic and liposomal preparation, for that Dayan & Touitou prepared a nanoethosomes that was loaded with trihexyphenidyl HCl and result that came were really great as nanoethosomes showed better transdermal permeation than both the above mentioned preparation [50]. The penetration of nanoethosomes was deeper as compared to conventional liposomes. Dubey et al. studied melatonin loaded nanoethosomes in cadaver skin of human and are compared with liposomes. Result showed high mobility of drug through skin in case of nanoethosomes.

Patent related to nanoethosomes
First patent related to nanoethosomes granted in 1986 by Prof. Elka Touitou of Hebrew University School of Pharmacy, Jerusalem. Descriptions of few patents regarding transdermal drug delivery of nanoethosomes are given in Table 5.

Conclusion
The work presented here is an overview on one of the current novel drug delivery device named as vesicular carrier. For researchers in the field of novel drug delivery vesicular carrier has become an area of interest which makes the future of vesicular carrier application bright in field of therapeutics and drug delivery. As vesicular carrier has an inert ability to better factors like solubility, penetration, uptake as well as it provides a better carrier facility to ensure the stability of various types of drugs and proteins, this makes it bankable delivery system of bioactive agents. In addition to this vesicular system is compatible with both hydrophilic as well as hydrophobic drug and easy encapsulate them. It also encapsulate herbal bio agent and ensure their stability. These said characteristics makes vesicular carrier system a promising option in the field pharmaceutical nanotechnology and herbal medicines. Vesicular carrier system has few thing as drawbacks like instability, relatively smaller short shelf life or difficulty to reach the target. All the drawbacks can be overcome by few techniques like providing appropriate storage condition to maintain stability, giving a polymeric coating to the preparation or conjugating the preparation with suitable ligands. To ascertain or assess the vesicular carrier stability and acceptability said parameters can be employed.  Table 5. Description of patents related to nanoethosomes for transdermal drug delivery.