Lipofection

Related Terms

Cationic liposome, chloroform, drug delivery, endocytosis, gene therapy, HIV, human immunodeficiency virus, lamellar structures, lipofection, liposomes, lysosomotropic agents, multilamellar, Myocet?, nonviral delivery, nucleotide, phagocytes, phosphatidylcholine, polyethylene glycol, REV, reverse-phase evaporation vesicle, targeted delivery, transfection, transformation, toxicity, unilamellar, vector, vesicles.

Background

General: Genes are considered the building blocks of life because they provide instructions for all the cells in the body. They are located inside cells and control an organism's development and functions by instructing cells to make new molecules. Genes are passed down from parents to their children.
Deoxyribonucleic acid (DNA) is a long thread-like molecule made up of large numbers of nucleotides. The sequence of bases in DNA serves as the carrier of genetic information. A chromosome is a long DNA molecule that carries the hereditary information of an organism.
Nucleotides are molecules composed of a nitrogen containing base, a 5-carbon sugar, and one or more phosphate groups. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T) in DNA. Long strands of nucleotides form nucleic acids. RNA (ribonucleic acid) is a nucleic acid that aids in protein synthesis, which is important for growth and maintenance of the body. RNA is formed under the direction of DNA. Both RNA and DNA help to form amino acids, which are the building blocks of protein.
Liposome: A liposome is a man-made small lipid (fat) vesicle that contains water or saline (salt solution) in the center. A vesicle is a small bag or pouch that is surrounded by its own membrane and is found inside the cell. The two-layered walls of the liposomes are made of phospholipids that are similar to the phospholipids found in the cell membrane. Depending on the number of lipid layers, liposomes may be classified as multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). MLVs consist of several lipid layers (up to 14) in an onion-like arrangement. SUVs and LUVs consist of a single lipid layer but differ in the diameter of the liposomes. SUVs are smaller in diameter (25-50nm), whereas LUVs range from 100nm up to 50 micrometers.
Liposomes were first produced by Alec D. Bangham in 1961. It was found that phospholipids combined with water and immediately formed a vesicle because one end of each molecule is water soluble, while the opposite end is not soluble in water (just like oil in water). Due to this ability, liposomes have been used as delivery vehicles for drugs, vaccines, or enzymes in the body. Nucleic acids of unlimited size can also be delivered, ranging from single nucleotides to large chromosomes. Additionally, different types of nucleic acids can be delivered, including DNA, RNA, oligonucleotides (short segment of DNA/RNA), viral nucleic acids, and several others.
Uses: The use of an efficient carrier for nucleic acid delivery is an important factor for the successful application of gene therapy. Liposomes have been used to carry normal genes into a cell in order to replace defective, disease-causing genes. This is known as gene therapy. Gene therapy is an experimental procedure that may help treat or prevent inherited disorders and some types of cancer. A genetic or inherited medical disorder is a condition that is caused by an abnormal expression of one or more genes. This occurs when the chemicals that make up an individual's genes are incorrectly deleted, added, or substituted (mutation). If the mutation causes the cells in the body to stop functioning properly, the person may develop a disease or disorder.
Liposomes have been shown to be efficient vehicles for many in vitro (a test performed in a test tube in laboratory) and in vivo (within the body) applications. Animal cells, plants cells, and bacteria are susceptible to transformation by liposomes. Transformation is the genetic alteration of a cell due to the incorporation or uptake of foreign genetic material. This alters the genetic makeup of the injected organism.
For example, genes representing characteristics such as drought resistance or pest resistance may be incorporated into plants. The use of liposomes for transformation or transfection is called lipofection. Transfection is the process of introducing foreign material into the cells of animals or plants using a viral vector or other methods of transfer. Vectors are carriers into which foreign genetic materials (DNA/RNA) are inserted. The vector containing the foreign DNA/RNAs is then introduced into a recipient or target cell and maintained for study or expression.
Advantages: Delivery of nucleic acids using liposomes is promising as a non-immunogenic (does not induce a specific immune response) approach to gene therapy and would overcome some of the disadvantages of viral vectors. The viral vector may cause toxicity or stimulate an immune and inflammatory response in the patient. There is also a slight chance that the virus may recover its ability to cause disease in the patient, even if researchers think they have removed all of its disease-causing genes. Also, in diseases affecting the nervous system, muscle movement, and pain (e.g. Parkinson's disease), viruses cannot be used as vectors because they are too big to make it across the protective membrane that separates circulating blood from brain cells (called the blood-brain barrier). Therefore, liposomes have been used as nonviral delivery vehicles. However, viral vectors have one advantage over the liposomal method; they have high efficiency (nearly 100%) in delivering the foreign material into the cell.

Methods

General: A liposome is a man-made small lipid (fat) vesicle that contains water or saline in the center. Saline is a salt solution, usually sodium chloride. A vesicle is a small bag or pouch that is surrounded by its own membrane inside the cell. The liposomes may be filled with drugs and delivered to cells to treat cancer and other diseases. In order for liposomes to be used commercially, the number of layers in the liposome needs to be predictable and the amount of drug in the carrier needs to be consistent from batch to batch.
Several methods are used to prepare the liposomes. Lipids of high quality (i.e., without any contaminants) should be used while preparing liposomes to achieve proper drug delivery. Some of the commonly used lipids for preparing liposomes are phosphatidylcholine (PC), dipalmitoylphosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine, sphingolipids, phosphatidylglycerol (PG), cardiolipin, glycolipids, gangliosides, and cerebrosides. Among these, phosphatidylcholine is preferred because it is readily available from various sources, such as soy plants, sunflowers, etc., at a minimal cost compared to other lipids. However, the preferred lipids depend on the characteristics required for the intended use of the vesicle. A combination of one or more lipids may also be used.
Generally, lipids or combinations of lipids will be either electrically neutral (no charge) or positively charged (cationic). Cationic liposomes are the preferred nonviral vectors because they easily interact with the DNA, which is negatively charged. Most of the lipids are commercially available with an organic solvent such as chloroform, acetone, and nail polish remover. A solvent is a liquid in which substances are dissolved in order to create a solution.
Conventional method: The conventional method was first described by Bangham and co-workers. In this method, the multilayered liposomal vesicles are prepared by the addition of biologically active materials (DNA, antibiotics, peptides, compounds that kill the pests) containing aqueous solution to a film of lipid. Aqueous solution is a solution wherein water is the solvent. The aqueous solution penetrates the film and hydrates the phospholipids. The hydrated phospholipids spontaneously come together to form vesicles. The major drawback of this method is that only low quantities of a drug can be entrapped because of the small surface area between the lipid film and the aqueous solution.
Modified method: A method was developed to increase the DNA or drug capture in liposomes by increasing the surface area between the lipid and the aqueous solution during the liposome membrane formation. In this method, water droplets containing the drug are surrounded by phospholipids in water and organic solvent emulsion. Emulsion is a suspension of small globules of one liquid in a second liquid with which the first will not mix, such as oil and water. The removal of the organic solvent forces the water droplets to come together, forming liposomes. This method is used to prepare single or oligo-layered vesicles (two or three layers) on a small scale because the amount of drug captured cannot be predicted beyond one or two layers.
Reverse-phase evaporation vesicle (REV) method: The REV method is an improved method of preparing many layered liposomes with high-capturing capacity. In this method, a lipid, organic solvent, water, and biological material are combined to form a mixture. Sound energy, such as ultrasound, is used to agitate/move the particles in the mixture (sonication) to give a water-in-oil emulsion. The organic solvent is removed by treating the mixture with inert gas, such as nitrogen or argon, or by evaporation under a reduced pressure in a vacuum to prevent the solvent from splashing. The removal of the organic solvent forces the conversion of lipid mixture to liposomal vesicles. The major advantage in this technique is that the amount of water-in-oil emulsion can be easily controlled, helping to determine the exact amount of biological material captured into the liposome and also the number of lipid bilayers in the liposome. Multilayered vesicles are formed when the emulsified aqueous phase is less in relation to the lipid content.
Liposomal delivery: The liposomes containing the biological materials may be made up into dosage forms such as injections, oral preparations, and suppositories, for easy delivery of the biological material into a chosen subject for treatment or for research purposes. A suppository is a small solid preparation shaped for ready introduction into one of the orifices of the body other than the mouth (such as the rectum, urethra, or vagina). It is made of a medicated substance, which is solid at room temperature but melts at body temperature. The route of administration of the liposomes may be directly into the vein, artery, muscle, brain, eye, or skin. Hence, it has the potential to be used anywhere in the body.
Entry into target cell: Endocytosis, the process wherein cells absorb molecules such as proteins from outside by engulfing them with the cell membrane, was widely believed as the major route by which the target cells uptake liposomes. Liposomes may interact with a cell membrane of the target cell in a way that permits the contents of the liposomes to enter the cytoplasm (gel-like material inside the cell membrane) of the cell. This interaction can take the form of complete or partial fusion of the membranes or cause localized disruptions of the cellular and liposomal membranes.
Another method by which liposomes enter the cell is with the help of monoclonal antibodies, which are produced from one type of immune cell that are derived from a single parent cell and target one type of antigen or substance. Monoclonal antibodies may be used to direct the DNA loaded liposomes to target cells because the antibodies recognize the surface antigen of target cells and specifically bind to it along with liposomes. An antigen is a substance that is capable of causing an immune response. Antibodies are proteins manufactured by the body that bind to an antigen to inhibit or destroy it. The cells are pretreated with compounds (lysosomotropic agents) that enter lysosomes, which are structures within the cell containing digestive enzymes. The lysosomotropic agents like chloroquine or colchicines are used to prevent the breakdown of liposomes by the digestive enzymes.
Detection: Transfection success and expression of the transfected nucleic acid in a cell can be detected in a number of ways. These generally depend on either detection of the physical presence of the nucleic acid in the cell, e.g., by including radionucleotides (nucleotide with a tracer) in the nucleic acid for easy detection or by detecting expression of the protein by techniques such as chemiluminescence method. This method uses a chemical dye that binds with a protein produced as a result of the transfected nucleic acid. The protein is attached to the probe or a sequence of DNA that can recognize and bind to the target of interest; it emits light that is detected by luminescent imager.

Research

General: Liposomal nonviral delivery vehicles have been used for various purposes, including gene therapy. Gene therapy involves inserting human genes into a patient in order to treat or prevent an illness. Below are some of the common disorders and diseases studied using liposomal nonviral delivery vehicles.
Parkinson's disease: Gene therapy has been studied as a possible treatment for Parkinson's disease, a degenerative nervous system disease. Researchers have recently found a way to transfer genes into the brain. Viruses cannot be used as vectors because they are too big to make it across the protective membrane that separates circulating blood from brain cells (called the blood-brain barrier). So, instead of using viral vectors, researchers have used liposomes that are coated in a polymer called polyethylene glycol (PEG) to deliver the new genes. This allows billions of copies of a gene to be inserted into the brain to calm overactive connections in the brain. Researchers are hopeful that this method may be an effective treatment for Parkinson's disease in the future.
Leukemia: Liposomes containing a standard anticancer drug combination (cytarabine and daunorubicin) have been studied both in vitro (a test performed in a test tube in laboratory) and in vivo (within the body) to treat leukemia. Leukemia is cancer of the bone marrow and blood that causes large numbers of abnormal blood cells to be produced. The medications penetrate the bone marrow and may help treat the cancer. The bone marrow is the soft, inner part of the bones that makes all types of blood cells. The liposomes enclosing the drugs have shown good response in both in vitro and in vivo studies.
Human immunodeficiency virus (HIV): Scientists are developing novel drug delivery systems in an effort to overcome the challenges associated with antiretroviral therapy (the standard treatment for HIV) to improve the management of HIV and AIDS (acquired immunodeficiency syndrome). Liposomal delivery system is one of the methods that has been studied to develop alternate routes of antiviral drug administration. Initial results have shown good response to the treatment.
Retinal diseases: Studies are being conducted to treat retinal (light sensitive part of the inside of eye) diseases by the delivery of the liposomal DNA complex to the retinal pigment epithelial (RPE) cells. The entry of the liposomal DNA into RPE was inhibited by the retina, but with the use of ultrasound (sound waves) energy, the entry of the liposomal DNA into the retina has been increased. Hence, injection of liposomes into the eye may be an effective tool to treat eye diseases in the future.
Liver diseases: Early human evidence suggests that the nutritional supplement S-adenosyl Methionine (SAMe) may normalize levels of liver enzymes in individuals with liver disease. Scientists have used liposomes containing SAMe to treat patients with liver failure and found some improvement. This was found to be due to the addition of a salt,1,4-butanedisulfonate salt (SD4), which improved the liposome SAMe formulation, suggesting that SD4 in liposomes may be a potential treatment for liver diseases.

Implications

General: Liposomal nonviral delivery vehicles are used in widespread applications and are currently used in human clinical gene therapy trials as well as in cell transfection applications for biological research. Transfection is the process of introducing foreign material into the cells of animals or plants using a virus vector or other methods of transfer.
Gene therapy: Gene therapy is an experimental procedure that involves inserting human genes into a patient in order to treat or prevent an illness. Liposomes have been used as effective delivery vehicles to treat several diseases such as cancer.
Drug-delivery system: Liposomes may be filled with drugs and used to deliver the drugs to treat cancer and other diseases. Liposomes help protect healthy cells from the toxicity of certain cancer drugs and help avoid the concentration of drugs in organs such as the liver and kidneys, thereby reducing or removing some of the common side effects associated with the drugs. Liposomes are effective in diseases that affect the phagocytes, the cells that ingest foreign agents like microorganisms. This is because liposomes accumulate in the phagocytes, which recognize them as foreign agents/invaders and engulf them.
Generally, liposomes are non-toxic, biodegradable, and non-immunogenic (do not produce a specific immune response). Associating a drug with liposomes markedly changes the process by which a drug gets absorbed, distributed, metabolized, and eliminated by the body and causes less harm to the entire body. Furthermore, the drug is prevented from early degradation and/or inactivation after the introduction to the target organism. Hence, liposomes are considered as efficient drug delivery vehicles to treat different diseases. Below are examples of commercial brands available using liposome as the delivery system.
Liposomal doxorubicin (Caelyx?, Myocet?): Liposomes may be used to deliver various types of drugs, including chemotherapy drugs. Liposomal doxorubicin is a chemotherapy drug used to treat different types of cancer such as breast and ovarian cancers. Chemotherapy drugs destroy cancer cells by preventing them from growing and multiplying. The doxorubicin molecules are enclosed within the liposome and given to the affected patients by a drip (infusion). The liposome allows the drug to remain in the body for a longer duration, facilitating the drug to be delivered to the cancer cells without destroying the healthy tissues (less toxicity).
AmBisome?: Liposomes containing amphoteracin B are used in the injection form to treat fungal infections caused by fungi such as Candida, Aspergillus, etc. This formulation of the drug in liposomes has helped to reduce the harm to the body, which commonly occurs when the drug is administered.

Limitations

One major drawback of earlier methods of liposomal nonviral delivery was low efficiency in transfection compared to viral vectors, especially in vivo (inside the body), thereby hindering their use for in vivo treatment applications. By making certain modifications/improvements, liposome nonviral delivery has achieved higher transfection efficiency. Transfection is the process of introducing foreign material into the cells of animals or plants using a virus vector or other methods of transfer. Vectors are carriers into which foreign genetic materials (DNA/RNA) are inserted.
The major drawback of the conventional method of liposomal preparation is that only low quantities of drug can be captured because of the small surface area between the lipid film and the aqueous solution, which are used during the liposomal preparation. This was overcome in methods that were developed later by making certain modifications.
The production of reproducible batches of liposome-DNA or other material complexes is a major challenge in the conventional method. This is because separate solutions of chemicals and DNA are mixed together resulting in a heterogeneous (different) mixture; for example, free liposomes and DNA may coexist within the same mixture containing liposomes enclosed with DNA. Also, conventional liposomal nonviral delivery method is dependent on the mixing procedure, composition of the reagents (chemicals that help the reaction process), and the time allowed for complex formation, causing difficulty in reproducing a similar batch every time.

Future research

General: Recent advances have dramatically improved transfection efficiencies and the usefulness of liposomal vectors. Researchers have demonstrated broad efficiency of a liposomal delivery system in small and large animal models for lung, breast, head and neck, and pancreatic cancers as well as for hepatitis B and C (liver infection). The liposomal delivery system is currently used in a clinical trial to treat non-small-cell lung cancer and will be used in upcoming clinical trials to treat other cancers and heart diseases. Transfection is the process of introducing foreign material into the cells of animals or plants using a virus vector or other methods of transfer. Vectors are carriers into which foreign genetic materials (DNA/RNA) are inserted. The vector containing the foreign DNA/RNAs is then introduced into a recipient or target cell and maintained for study or expression.
Intranasal vaccines: Researchers are conducting studies using liposomal nonviral delivery vehicles to deliver vaccines through the nasal route. Although the initial results show promise, further research is required to prove the safety and effectiveness of nasally delivered vaccines. Vaccines contain small amounts of disease-causing organisms that allow the immune system to produce antibodies to the foreign invader; consequently, individuals become immune to the specific illness after receiving a vaccine.
Anti-jet lag agent: Jet lag is a disturbance in an individual's internal clock, called circadian rhythm, due to long distance travel with time changes. Few studies have suggested that melatonin may help to avoid jet lag if taken on the day of travel and continued every 24 hours for several days, while others indicate that it has to be taken 3-4 days before the travel. Hence, more trials are being carried out to determine the exact dosing of melatonin. Melatonin is a hormone produced by the body that regulates the sleep cycle. Scientists are conducting in vitro studies using liposomes containing melatonin through the skin to reduce jet lag. Further larger trials are required to examine this method.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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