Different approaches for delivering the drug through vesicular carriers

http://dx.doi.org/10.21276/IJRDPL.22780238.2018.7(4).3071-3082 ABSTRACT:Delivering the drug molecule through conventional ways of drug delivery has been a challenging task for the researchers since years. Many novel technologies and system have been developed to overcome this limitation. Vesicular carriers such as niosomes, liposomes, aquasomes, phytosomes, etc. have been developed as one of the novel ways for the delivery of drug molecules effectively. They provide various advantages such as increased bioavailability, increased pharmacological activity, decreased toxicity, enhancement of stability, sustained delivery, and protection from physical and chemical degradation that occurs inside the body over conventional delivering systems. These vesicular systems can be lipidic or nonlipidic. The composition of niosomes involves nonionic surfactantslipids and liposome involves phospholipids (natural and synthetic). This review article describes the study of the four vesicular carriers namely niosomes, liposomes, aquasomes and phytosomes and their various aspects such as their definition, types, methods of preparation, advantages, disadvantages, evaluation and characterization. Such different systems are widely used in gene delivery, tumor targeting to brain, oral formulations, in stability and permeability problems of drugs.


INTRODUCTION
The application of vesicular system in drug delivery has changed the definitions of diagnosis and treatment in different aspects of biomedical field. The vesicular system such as liposomes, niosomes, aquasomes, and phytosomes are used to improve the therapeutic index of both existing and new drug molecules by encapsulating an active drug inside vesicular structure in one such system.
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. 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.

Materials used for the preparation of aquasomes
The following materials are used for the preparation-1. Core material-Ceramic like diamond particles, brushite (calcium phosphate) and tin oxide are used. Polymers can also be used as core materials.
3. Bioactive/ drug layer-The drug is coated and has a property of interacting with coating film by means of non-covalent and ionic interactions. [2,3,4] The method of preparation involves 3 steps-A. Formation of an inorganic core-1) Tin oxide-It can be synthesized by using a highpressure mixture of argon and oxygen, on which 3-inch diameter of high purity tin is sputtered. As a result ultrafine particles are formed in the gas phase, which are collected on copper tubes cooled to 770k with flowing nitrogen.

Method of Preparation of aquasomes
2) Brushite (calcium phosphate) core-A solution of disodium hydrogen phosphate is reacted with calcium chloride to form calcium phosphate.
2Na2HPO4 + 3CaCl2 +H2O  Ca3(PO4)2 + 4NaCl + Cl2 + [O] (Disodiumphosphae)(Calciumhydrogen phosphate) The precipitated calcium phosphate core particles are centrifuged and washed with distilled water to remove sodium chloride. To obtain the core particles of desired size, the precipitate is resuspended in distilled water and passed through membrane filter.
3) Diamond core-Diamond particles are subjected to ultra-cleansing followed by sonification to form the inorganic core.

B. Coating of core
The coating material is added to the aqueous dispersion of core under sonication. The coated core is subjected to lyophilization, which helps to make an irreversible adsorption of coat on the core surface. Then by centrifugation, unabsorbed coating particles are removed.

C. Loading of drug layer
In this stage, a solution of known concentration of drug is prepared in a suitable pH buffer and coated particles are dispersed into it. The dispersion is kept overnight at low temperature, which governs drug loading after some time to obtain drug-loaded formulation.
Evaluation of aquasomes [7] a. Size and shape -The mean particle size and size distribution are determined by a photon correlation spectroscopy using aAutosizer II C apparatus and SEM. f. The antigen-loading efficiency for the aquasomes-Accurately weight antigen-loaded aquasome formulations were suspended in Triton X-100 and incubated in a wrist shaker for 1 h. Then, samples are centrifuged at and absorbance is determined using micro-BCA methods with set a blank of unloaded aquasomes formulation. Antigen loading is expressed as per unit weight of aquasomes particles (g of antigen/mg of sample).

Characterization of Aquasomes
a) Size distribution-Particle size distribution and morphological properties can be determined by SEM (Scanning electron microscope) and TEM (Transmission electron microscope). For measuring mean particle size and zeta potential of particle, photon correlation spectroscopy is used. b) Structural analysis-FTIR Spectroscopy using potassium bromide pellets is performed for structural determination. IR is recorded in wave number range 4000 -400cm-1.
c) Crystallinity-X-Ray diffraction is used to determine crystalline/amorphous behavior of ceramic core. d) Coating elegance and strength-Concanavalin Ainduced aggregation method or anthrone method is used for determining coating strength and adsorption of coating on core. e) Glass transition temperature-The transition from glass to rubber state as a change in temperature upon melting is recorded by DSC (Differential Scanning Calorimetry).
f) In vitro drug release study-The release pattern of drug from aquasomes is determined by incubating a known quantity of drug-loaded aquasomes in incubator at 370C. The sample is withdrawn at regular intervals and centrifuged at high speed and estimated for drug content by UV Spectroscopy.
Applications of aquasomes [7] 1. Aquasomes are used for delivering of enzyme like DNAs and pigments/dyes.
2. Aquasomes can be used for pharmaceutical delivery like insulin.
3. They have been used for successful targeted intracellular gene therapy.
4. They are used as red blood substitutes.
5. They are used as vaccines for delivery of viral antigen i.e Epstein-Bars virus, Immunodeficiency virus antigen to evoke the correct antibody.

PHYTOSOMES
The term phyto means plant and some means cell like bodies. Phytosomes are patented methods developed by Indena to include phospholipids into herbal extracts. Phytosome is a newly introduced patented technology developed to incorporate standardized plant extracts or water-soluble phytoconstituents into phospholipids to produce lipid compatible molecular complexes, which enhances their absorption and bioavailability.
The phospholipid used commonly is phosphatidylcholine. Phosphatidylcholine is a bifunctional compound in which the phosphatidyl moiety is lipophilic and choline moiety is hydrophilic in nature. Lipophilic moiety forms the tail and hydrophilic moiety (choline) forms the polar head.
The drug is incorporated as an integral part of membrane into the polar head part of phospholipid (phosphatidylcholine) by means of chemical bonds.
The Phytosome technology produces a little cell, better able to transit from a hydrophilic environment into the lipid-friendly environment of the enterocyte cell membrane and from there into the cell, thus protecting the valuable components of the herbal extract from destruction by digestive secretions and gut bacteria. [8,9,10]

Differences between liposomes and phytosomes -
Phytosomes differs from liposomes in the fact that, drug in phytosomes remains chemical bonded to the polar head part of membrane, while in liposomes drug is dissolved within the cavity or may remain in membrane. [11] Method of preparation of phytosomes [12] 1. Solvent evaporation method-A fixed quantity of drug, polymer and phospholipid (phosphatidylcholine) is taken in a round bottom flask (RBF). It is refluxed with specific solvent at 50-600C for 2 hours. Then the mixture is concentrated to 5-10 ml to get the precipitate, which is filtered and collected. The dried precipitate is phytosome loaded with drug. 2. Rotary evaporation technique-Quantity of drug, polymer, and solvent is taken in a rotary flask and stirred for 3 hours below 400C. As a result, thin film of sample is obtained to which n-Hexane is added and continuously stirred using magnetic stirrer. As a result, phytosomes loaded with drug are obtained, that are collected and stored at room temperature. 3. Antisolvent precipitation technique-Known quantity of drug, phospholipids and polymer is taken in round bottom flask (RBF) and refluxed with specific solvent not exceeding 600C for 2 hours. The mixture is concentrated to 5-10ml and n -Hexane is carefully added to it with continuous stirring to get the precipitate, which is filtered and kept in vacuum desiccator overnight. The dried precipitate is crushed in mortar and sieved through 100mesh size. As a result, phytosomes loaded with drug are obtained, which are placed in amber coloured glass bottles at room temperature.
Properties of phytosomes [13,14] 4. Physico Chemical properties-Phytosomes is a complex between a natural product and natural phospholipids, like soy phospholipids. Such a complex is obtained by reaction of stoichiometric amounts of phospholipids and the substrate in an appropriate solvent. The main phospholipids substrate interaction is due to the formation of hydrogen bonds between the polar head of phospholipids (i.e. phosphate and ammonium groups) and the polar functionalities of the substrate. When treated with water, phytosomes assumes a micellar shape forming liposomal like structures. 5. Biological properties-Phytosomes are better absorbed, utilized and as a result produce better results than conventional herbal extracts. The increased bioavailability of the phytosome over the non-complexed botanical derivatives has been demonstrated by pharmacokinetic studies or by pharmacodynamic tests.

Advantages of phytosomes
 Phosphatidylcholine, component of phytosome acts a carrier and gives hepatoprotective effect.
 The composition of phytosome is safe.
 The absorption and bioavailability of water-soluble phytoconstituents is increased. This results in better therapeutic effects.
 Due to increased bioavailability of phytoconstituents the dosage required to produce desirable effect is reduced.
 The phytosomes have a better stability than liposomes.
 Phospholipids add to the nutritional value of the plant extract.
 High market demand for products.
 The process of manufacturing phytosomes is relatively simple.
 Phytosomes have the ability to permeate through skin.

Disadvantages of phytosomes
 Phytoconstituent is rapidly eliminated from phytosomes.
 The duration of action is short.

Method of evaluation of phytosomes
1) Percentage yield-It is calculated by the following formula -% Yield= Practical Yield * 100 Theoretical Yield 2) Differential scanning calorimetry (DSC)-Glass transition temperature is noted by DSC.

4)
Particle size-It is determined by Zetasizer Zen 3600 at a fixed scattering angle of 90 degree at 25oC.

5) Entrapment efficiency-It is determined by following formula-
Entrapment efficiency (%) = (Total amount of drug -Amount of free drug) * 100 Total amount of drug To determine total amount of drug, 0.1 ml phytosome loaded suspension is diluted upto 10ml and estimated with UV Visible Spectrophotometer.
To determine amount of free drug, 0.1 ml phytosome loaded suspension is diluted upto 10ml solvent and centrifuged at 18000 rpm for 30 min at -40C using cooling centrifuge machine. The supernatant is isolated, and quantity of free drug is estimated by UV Visible Spectrophotometer.

Transition temperature:
The transition temperature of the vesicular lipid system can be settled via differential scanning calorimetry.

Entrapment efficiency:
The entrapment efficiency of a phytosomal preparation can be determined by exposing the preparation to ultracentrifugation method.
3. Vesicle size and zeta potential: The particle size and zeta potential of phytosomes can be confirmed by dynamic light scattering, which usages a computerized examination system and photon correlation spectroscopy.

Surface tension activity measurement:
The surface tension activity of the drug in aqueous solution can be determined by the Du Nouy ring tensiometer. When large numbers of phospholipids are placed together they will arrange themselves spontaneously to match their heads together on one side and tails on the other side. Under specific physical conditions concentric bilayered vesicles are formed (means 2 layers of phospholipids make concentric vesicles). As a result a hollow sphere is formed in which aqueous molecules can be entrapped.  [16,17,18,19,20,21,22,23] 1) Lipid Hydration Method (Used mainly for preparing MLVs and OLVs)-In this method lipids are dissolved in suitable organic solvent. This organic solution of lipid is placed in a flask or beaker and dried to form a thin film at the bottom of vessel. The thin film is hydrated by adding an aqueous buffer (containing water soluble drug). Heat liposomal dispersion at 600C for 10 min to obtain liposome. It is agitated to encapsulate the drug in lipid bilayer film. The dispersion of MLVs is passed through a microfluidizer at very high velocity. They reach in the precisely defined microchannels present in the interaction chamber of microfluidizer. Here pressure is very high upto 10,000 psi. Thus LUVS are obtained. The reduction in the size range can be achieved by recycling of the sample. The process is reproducible and yields liposomes with good aqueous phase encapsulation.

7)
Freeze-Thaw Method: The method of freezing and thawing is introduced for increasing the trapped volume of liposomal preparations. The freeze-thaw method is dependent on the ionic strength of the medium and the phospholipid concentration. It influences to a physical disruption of lamellar structure leading to formation of unilamellar vesicles. The unilamellar vesicles are rapidly frozen followed by slow thawing, while the freeze and thawing cycles are repeated. The preparation of MLV propranolol liposomes by freeze-thaw method is described in the literature. The liposomal propranolol formulation is prepared by using distearoylphosphatidylcholine and dimyristoylphosphatidylcholine as phospholipids in phosphate buffered saline buffer, followed by six freeze-thaw cycles 8) Calcium-Induced Fusion Method: The calcium-induced method is based on adding of calcium toSUV. On adding calcium to a dispersion containing SUVs, the individual SUVs get fused to form vesicles inspiral configuration. When EDTA is injected to this, it results in formation of LUVs.

Advantages of liposomes
1) Liposomes encapsulated drugs are delivered intact to cells and tissues and can be released when liposome bilayer is destroyed. Thus, it enables site specific and targeted drug delivery.
2) Liposomes can be used for both hydrophilic and lipophilic drugs without chemical modification.
3) Other tissues and cells of body are protected from the drug until it is released by liposome, thus toxicity does not occur.
4) The size of liposome can be altered depending on the nature and dose of drug as well as an intended use of product.
5) They are biocompatible, have high stability and high diffusivity in the skin.

Disadvantages of liposomes
 Less stability Amphotec-It is a sterile lyophilized powder meant for intravenous administration that is to be reconstituted before use.

Daunoxome-
It is liposomal vesicles composed of an aqueous solution of daunorubicin citrate encapsulated with membrane of distearoylphosphatidylcholine and cholesterol (2:1). This liposomal formulation helps to protect Daunorubicin from chemical and enzymatic degradation, minimizes protein binding and decreases its uptake by normal tissues.

Doxil-
It is a liposome made of dauxorubicin-HCl encapsulated in lipid membrane made of phosphoethanolamine sodium, phosphatidylcholine, cholesterol lipids and ammonium sulphate with histidine as buffer. Methotrexate has also shown increased effectiveness in form of liposomes.

3) Targeting of antiviral drugs-Ganciclovir and Foscarnet
are the antiviral drugs that are used in CMV retinitis. CMV retinitis is a disease of retina caused due to Cytomegalovirus. It leads to blindness. These drugs are given by IV injection but have narrow therapeutic index and low safety margin. Ganciclovir causes bone-marrow depression and Foscarnet causes renal (kidney) damage.
To reduce these adverse effects, Ganciclovir and Foscarnet are prepared as liposomes with phosphatidyl ethanolamine-cholesterol lipids 4) Site specific delivery of antibiotics-Gentamycin is an aminoglycoside antibiotic obtained from micromonosporapurpurea. It is used in respiratory tract infections, pneumonia, lung abscess, but it causes ototoxicity and nephrotoxicity, which can be reduced by preparing its liposomes using phosphatidylcholine. Niosomes are microscopic lamellar structures. They have a bilayer made up of non-ionic surfactant. The bilayer has its hydrophilic ends exposed on the outside and inside of the vesicle, while their hydrophobic tails face each other within the bilayer. Thus, the vesicles are formed where polar drugs can be entrapped, while hydrophobic drugs can be incorporated in the bilayer tail region itself.
Advantages of niosomes [26] 1) Niosomes are highly stable structure. They require no special conditions such as low temperature or inert atmosphere for storage.
2) They can entrap both hydrophilic and hydrophobic drugs.
3) They are non-toxic, biodegradable, biocompatible, and non-immunogenic.  [27,28,29,30,31,32,33] 1. Ether injection method -Nonionic surfactant with or without a small amount of cholesterol to improve stability is dissolved in diethyl ether. This solution is injected slowly into an aqueous drug solution at 600C through a 14-gauze needle. Ether is vaporized by rotatory evaporator to form SUVs.

3.Sonication Method-
In this method an aliquot of drug solution in buffer is added to the surfactant/cholesterol mixture in a 10ml glass vial. The mixture is probe sonicated at 60°C for 3 minutes using a sonicator with a titanium probe to yield niosomes.  Zeta potential analysis-Zeta potential analyzer based on electrophoretic light scattering and laser Doppler velocimetry method.

Micro fluidization Method-
 Scanning electron microscopy -Particle size  Optical Microscopy -viewed under a microscope  Measurement of vesicle size -Laser diffraction particle size analyzer  Entrapment efficiency-Drug entrapped in niosome is determined by complete disruption using 50% npropanol or 0.1% Triton-X-100 and analyzing by UV.
Entrapment efficiency (EF)= (Total amount of drug entrapped) * 100 Separating the unentrapped drug by dialysis centrifugation or gel filtration, drug remained entrapped in niosomes is determined by complete vesicle disruption.
 Vesicle diameter-It can be determined by electron microscope (or optical microscope)  In vitro release-It is performed using dialysis tube. A dialysis sac is washed and soaked in distilled water. The vesicle suspension is placed into dialysis bag and sealed. It is placed in 200ml Phosphate buffer in a 250 ml beaker with constant shaking at 37 degree Celsius. At continuous interval 5 ml sample is withdrawn and analyzed for drug content by UV. [39] 1. Delivery of peptide drugs-Peptide drugs when given orally as such are broken down by GIT enzymes resulting in loss of drug. This has been substantially reduced by using niosomal vesicles of peptide drug. For example, oral delivery of vasopressin derivative in niosomal vesicle significantly improved its stability.

Applications of niosome
2. Niosomes as a carrier for Hemoglobin-Niosomal suspension can be used as a carrier for hemoglobin. Niosomal vesicles are also permeable to oxygen. So, act as a carrier for hemoglobin in anemic patient.
3. Transdermal delivery of drugs by niosomes-Niosomes enhances the penetration of drugs through the skin. The niosomal technology has been successfully used in topical administration of antibiotics for treatment of skin diseases.

Antineoplastic Treatment-Antineoplastic drugs like
Doxorubicin and Daunorubicin causes severe adverse effect like cardiotoxicity and methotrexate causes bonemarrow depression and megaloblastic anaemia but niosomal entrapment of these drugs decreases tumor proliferation rate and enhances the plasma drug conc. in tumor cells only, thus reducing the toxicity on normal cells.

5.
Ophthalmic drug delivery-Bio adhesive-coated niosomal formulation of acetazolamide prepared from span 60, cholesterol stearylamine or dicetyl phosphate exhibits more tendencies for reduction of intraocular pressure.
6. Localized Drug Action-Due to their size and low penetrability through epithelium and connective tissue keeps the drug localized at the site of administration. Localized drug action results in enhancement of efficacy, potency of the drug and at the same time reduce its toxic effects.