Reverse Phase Evaporation Method Biology Essay


Swine flu is a kind of a respiratory disease which is common in pigs. The H1NI virus contains a combination of gene segments from a human, swine and avian influenza A viruses. This virus was first appeared in Mexico. As, this virus is a combination of different flu viruses, the H1N1 flue is much harder to treat. Typical flu viruses include the potential to kill more than 350000 victims. Season after season this disease seems to appear, sometimes showing mild symptoms and sometimes severe. Children, pregnant women and young adults appear to be the most affected1.

Swine flu is real cause for worry and many have been affected with it in the recent times. The infection spreads from one person to another like other common ailment such as cold or cough. Therefore the prevention is better than cure. When it is detected, treatment should be a priority. Even Physicians often prescribe immediate drug therapy for preventing more harm to the affected. Therefore taking proper medication treatment will always safeguard the affected person. The treatment for swine flu may last for 10-15 days under the medication of physicians. It is always safe to be under supervision of physicians until they confirm that the patient is free from virus1.

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Treatment for swine flu can be very effective. It is important to give plenty of rest to the patient in a ventilated room with warm interiors. Pain relievers should be given and fluid intake is suggested. Drinking of warm liquids is also suggested by the physicians. The patients with other diseases such as diabetes, heart disease and other ailments should take more care when they affected with H1N1 virus.  Old age people who are above the age of 65 are at a high risk when affected with swine flu, because the immune system of old age people works slowly when compared to adults. Maintaining an excellent personal hygiene system such as by hand wash, face masks, gloves are some of the important aspects for preventing swine flu. Those who are affected with swine flu must take extra care in personal hygiene level apart from good diet and complete rest for few days and should definitely offer relief for the person.

The fear that is spreading about swine flu is incorrect and such a large extent of phobia is not required, as there are excellent vaccines and medication available now for treatment of H1N1. It is also stated that by keeping the body oxygenated, one can safeguard and protect from swine flu.  The virus is very much similar to the mosquito byte and therefore, if the body is oxygenated, virus cannot attack the body2.

1.1.2 DRUG

Oseltamivir is indicated for the treatment and prevention of infections due to influenza A and B virus l3.

Mechanism of action: The prodrug Oseltamivir is itself not virally effective, however after administration into body it reaches the liver and there it is converted by natural chemical processes, hydrolyzed hepatically to its active metabolite, the free carboxylate of Oseltamivir. Oseltamivir is a neuraminidase inhibitor, serving as a competitive inhibitor towards sialic acid, found on the surface proteins of normal host cells. By blocking the activity of the viral neuraminidase enzyme, Oseltamivir prevents new viral particles from being released by infected cells3.

Dose: Marketed under the trade name Tamiflu, as capsules - 75 mg and as a powder for oral suspension-12 mg/ml.

Side Effects: The most common side effects reported in patients receiving Oseltamivir during studies for the treatment of influenza were nausea, vomiting, bronchitis, insomnia, and vertigo. Nausea and vomiting were generally mild to moderate3.


Liposomes are composite structures made of phospholipids contain small amounts of other molecules. The size of the liposomes ranges from 20 nm to 10 nm in diameter. However, for injectables all liposome formulations are in the submicrone ultrafilterable range (200 nm) size and can be considered as "Nanosize Particulates". Liposomes were first discovered by British haematologist Dr. Alec D. Bangham, FRS in 1961 (published 1964), at the Babraham institute, Cambridge4.

Fig no:1. liposome structure13

Main advantages of liposomes are that they offer drug delivery by increasing therapeutic index and greatly reducing the side effects. Liposomes are potential carrier for controlled drug release of tumours therapeutic agents and antibiotic, for gene and antisense therapy through nucleic acid sequence delivery, immunization through antigen delivery and for antiparkinsons. Last one decade, pharmaceutical researchers use the tools of biophysics in evaluating liposomal dosage forms. Liposomes have brought predominant changes in medical and some non-medical areas like bioreactors, catalysts, cosmetics and ecology.

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There are number of the structural and nonstructural components of liposomes, major structural components of liposomes are


Phospholipid is the major component of the biological membrane; two types of phospholipids are used natural and synthetic phospholipids. The most common natural phospholipid is the phospatidylcholine (PC) is the amphipathic molecule and also known as lecithin. It is originated from animal (hen egg) and vegetable (soyabean)5.


Incorporation of cholesterol in liposome bilayer can bring about big changes in the preparation of these membranes. It does not mean by itself form bilayer membrane structure, but can be incorporated into phospholipids membrane in very high concentration up to 1:1 or 2:1 molar ratios of cholesterol to phospatidylcholine5.


To understand the performance characteristic of liposomal systems, it is important to understand the mechanism of introducing drugs into liposomes. This is achieved by three primary mechanisms: encapsulation, partitioning and reverse loading6.


The physicochemical properties of the drug itself, especially solubility and partition coefficient, are important determinant of the extent of its incorporation in liposomes. It is useful for water-soluble drugs (doxorubicin, penicillin G) the encapsulation is simple hydration of a lipid with an aqueous solution of drug. The formation of liposomes passively entraps dissolved drug in inter lamellar spaces, essentially encapsulating a small volume6.


A drug substance that is soluble in organic solvents (cyclosporine) will go through partitioning. It is dissolve along with phospholipid in a suitable organic solvent. This combination is dried first after than added directly to the aqueous phase and solvent residues remove under vacuum. The acryl chains of the phospholipids provide a solubilizing environment for the drug molecule. This will be located in the intra bilayer space6.

Reverse loading

The reverse-loading mechanism uses for certain drugs (5-fluorouracil, Mercaptopurine) may exist in both charged and uncharged forms depending on the pH of the environment. This type of drug can be added to an aqueous phases in the uncharged state to permeate into liposomes through their lipid bilayers. Then the internal pH of the liposome is adjusted to create a charge on the drug molecules. Once, charged the drug molecules no longer is lipophilic enough to pass through the lipid bilayer and return to the external medium6.

Advantages of liposomes7

Liposomes have been widely used for many years in the pharmaceutical industry for

Controlled release and transfer of drugs and more recently by the cosmetic industry.

Versatile liposomes have been used in cancer therapy, gene manipulations.

A special quality of liposomes is that they enable water soluble, and water insoluble materials to be used together in a formulation without the use of surfactants and emulsifiers.

Long-circulating forms for specific targeting of the tumours and Superior drug release at target site.


More difficult to synthesize

More immunogenic

Limited target availability


The main aim and objective of the study is to prepare and develop controlled release liposomes for the treatment of swineflu.

To improve patient compliance.

To decrease the dosage frequency.

To increase rate of bioavailability.

To achieve a good release at the targeted site.



a) Handshaking Method

In order to produce liposome lipid molecules must be introduced into an aqueous environment. When dry lipid layer film is hydrated the lamellae swell and grow into myelin figures. Only mechanical agitation provided by vortexing, shaking, swering or pippeting causesmyelin figures to break and reseal the exposed hydrophobic edges resulting in the formation of liposomes can be made by hand shaken method8.

b) Sonication Method

This method is probably the most widely used method for the preparation of small Unilamellar vesicles. There are two sonication techniques:

Probe Sonication

The tip of sonicator is directly immersed into the liposome dispersion is very high in this method. The dissipation of energy at the tip results in local overheating and therefore the vessel must be immersed into an ice bath. During the sonication up to one hour more than 5% of the lipids can be de-esterify. Also, with the probe sonicator, titanium will slough off and contaminate the solution.

Bath Sonicator

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The liposome dispersion in a tube is placed into a bath sonicator. Controlling the temperature of the lipid dispersion is usually easier in this method compare to sonication the dispersion directly using tip. Material being sonicated can be kept in a sterile container, unlike the probe units, or under an inert atmosphere. The lipid bilayer of the liposomes can fuse with other bilayers, thus delivering the liposome contents. By making liposomes in a solution of DNA or drug they can be delivered past lipid bilayer9.

c) Reverse Phase Evaporation Method

Historically this method provided a breakthrough in liposome technology, since it allowed for the first time the preparation of liposomes with a high aqueous space-to-lipid ratio and able to entrap a large percentage of the aqueous material presented. Reverse phase evaporation is based on the formation of inverted micelles. These inverted micelles are formed upon sonication of a mixture of a buffered aqueous phase, which contains the water soluble molecules to be encapsulated into the liposomes and an organic phase in which the amphiphilic molecules are solubilized. The slow removal of the organic solvent leads to transformation of these inverted micelles into a gel like and viscous state. At a critical point in this procedure, the gel state collapse and some of the inverted micelles into a gel like and viscous state. At a critical point in this procedure, the gel state collapse and some of the inverted micelles disintegrate. The excess of phospholipids in the environment contributes to the formation of a complete bilayer around the remaining micelles, which results in formation of liposomes. Liposome made by this method can be made from various lipid formulations and have aqueous volume to lipid ratios that are four time higher than multi lamellar liposomes or hand shaken method10.

d) Freeze Dried Rehydration Method

Freeze dried liposomes are formed from preformed liposomes. Very high encapsulation efficiencies even for macromolecules can be achieved using this method. During the dehydration the lipid bilayers and the material to be encapsulated into the liposomes are brought into close contact. Upon reswelling the chances for encapsulation of the adhered molecules are much higher. The rehydration is a very important step and is should be done very carefully. The aqueous phase should be added in very small portions with a micropipette to the dried materials. After each addition the tube should be vortexed thoroughly. As a general rule the total volume used for rehydration must be smaller than the starting volume of the liposome dispersion11.


Several formulations will be prepared with different concentrations of above category of ingredients and all these formulations will be characterized for their physico-chemical and pharmacokinetic characteristics. The drug release studies will be performed by using in vitro methods such as dissolution studies using external membrane, content uniformity, Surface morphological studies using Scanning Electron Microscopy and Atomic Force Microscopy, Drug entrapment studies and ex vivo methods. Those formulations exhibiting optimum release profile will be studied for in vivo release studies using suitable animal models. Pharmacokinetic studies such as Tmax, Cmax, t1/2 , AUC and elimination rate constant will be determined after the formulation is given to the suitable animal models. Stability studies will be carried out as per ICH guidelines. Selected formulations will be studied for skin irritation, acute and chronic toxicity studies12.


Since the discovery of liposomes or lipid vesicles derived from self forming enclosed lipid bilayers upon hydration, liposome drug delivery systems have played a significant role in reformulation of potent drugs to improve their therapeutics. This has facilitated the application of a wide range of liposomal drugs in the treatment and prevention of diseases in experimental animals and clinically. The flexibility in their behavior can be exploited for drug delivery through any route of administration and for any drug or material irrespective of its physicochemical properties. Commercial introduction of the various liposomal formulations represents a milestone in the history of liposomal drug delivery. Many more liposome-based drug formulations can be expected in the near future both for delivery of conventional drugs and for new biotechnology therapeutics such as recombinant proteins, antisense oligo nucleotides and cloned genes.