Depend on the number of bilayers liposomes can be divided into multilamellar and unilamellar vesicles. According to the size unilamellar vesicles can be further divided into SUV LUV, GUV. They are prepared by different methods. SUV smaller than 100 nm diameter by sonication, LUV 100 nm to 1micro meter can be prepared by extrusion GUV larger than 1 micro meter can be prepared by evaporation.
SUV are typically 15-30nm in diameter while LUV range from 100-200nm or larger. LUV are stable on storage, however, SUV will spontaneously fuse when they drop below the phase transition temperature of the lipid forming the vesicle.
There are many methods to characterize the liposomes. To characterize the morphology of liposomes EM LS are the most common method. I will shortly discribe EM and LS here the details can be found in literature.
Liposome: Novel Drug Delivery System Jignesh Patel M. Pharm.(sem-3) NPC,visnagar
<ul><li>Lipids, along with proteins and nucleic acids, are essential biomolecules for the structure and function of living matter </li></ul>
What is a liposome? Liposomes are spherical self-closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent into their interior. The size of a liposome ranges from some 20 nm up to several micrometers and they may be composed of one or several concentric membranes, each with a thickness of about 4 nm. Liposomes possess unique properties owing to the amphiphilic character of the lipids, which make them suitable for drug delivery. Formation of such a typical structural configuration is attributed to the amphiphilic character of phospholipids. When the letter are dispersed in excess of an aqueous phase, hydration of the polar head groups of the lipid results in a heterogeneous mixture of closed structures.
What is a liposome? <ul><ul><li>Spherical vesicles with a phospholipid bilayer </li></ul></ul>Hydrophilic Hydrophobic
Fundamental properties: <ul><li>1. Size: </li></ul><ul><li>SUV: Small unilamellar vesicles 0.02-0.05 µ </li></ul><ul><li>LUV: Large unilamellar vesicles 0.1-0.5 µ </li></ul><ul><li>MLV: Multilamellar vesicles 1.0- >10 µ </li></ul><ul><li>2. Fluidity: </li></ul><ul><li>3. Surface properties: </li></ul>
Size determined by methods: Sonication: SUV Smaller than 100 nm diameter Extrusion: LUV (Size depends on the filters) 100 nm—1 µm diameter Evaporation: MUV Larger than 1 µm diameter
Materials used for preparation: <ul><li>Natural Phospholipids </li></ul><ul><li>Synthetic Phospholipids </li></ul><ul><li>Sphingolipids </li></ul><ul><li>Steroids </li></ul><ul><li>Polymeric materials </li></ul><ul><li>Charge lipids </li></ul>
Phospholipids: Polar Head Groups Three carbon glycerol
<ul><li>Each phospholipid </li></ul><ul><li>includes </li></ul><ul><li>a polar region: glycerol , carbonyl of fatty acids, P i , & the polar head group ( X ) </li></ul><ul><li>2 non-polar hydrocarbon tails of fatty acids ( R 1 , R 2 ). </li></ul><ul><li>Such an amphipathic lipid </li></ul><ul><li>may be represented as at </li></ul><ul><li>right. </li></ul>3D picture of a Phospholipid
<ul><li>Selection of bilayer component is imp from toxicity as well as shelf life optimization point of view. </li></ul><ul><li>Common process steps for liposome formulation: </li></ul><ul><li>Bilayer components are mixed in a volatile organic solvent or solvent mixtures (eg. Chloroform, ether, EtOH, or combination of these) </li></ul><ul><li>Cholesterol upto 40-50 mol % is included to provide greater stability in biological fluids. </li></ul><ul><li>A charged species may be added (5-20%) to prevent aggregation. (natural acidic phospholipids eg PS, PG, PI, PA, etc) </li></ul><ul><li>Small amt of antioxidants such as aplha-tocopherol are included when polyunsaturated natural lipids are used. </li></ul><ul><li>Once a suitable solution of the lipid component is made, the mixture may be filtered to remove minor insoluble components or ultra filtered to lower pyrogens. </li></ul><ul><li>The solvent is subsequently removed under conditions (Pressure and Temp) that ensure no phase separations of the component of the mixture take place. </li></ul>
<ul><li>Preparation of lipid for hydration: </li></ul>
Methods to check the morphology of liposomes: <ul><li>Freeze-fracture electron microscope (FFEM) or electron microscope (EM) </li></ul><ul><li>Light scattering (LS) </li></ul>
Methods to control vesicle size of liposomes: <ul><li>Centrifugation </li></ul><ul><li>Size-exclusion chromatography </li></ul><ul><li>Homogenization </li></ul><ul><li>Extrusion </li></ul>
<ul><li>2. Chemical Parameters: </li></ul><ul><li>Quantitative determination of phospholipid </li></ul><ul><li>Phospholipid hydrolysis </li></ul><ul><li>Phospholipid analysis </li></ul><ul><li>Cholesterol analysis </li></ul>
Classes of liposomes: <ul><ul><li>Conventional </li></ul></ul><ul><ul><li>Long circulating </li></ul></ul><ul><ul><li>Immuno </li></ul></ul>Cationic
Modes of liposome/cell interaction: Adsorption Endocytosis Fusion Lipid transfer
Uses of liposomes: Chelation therapy for treatment of heavy metal poisoning Enzyme replacement Diagnostic imaging of tumors Study of membranes Cosmetics Drug Delivery
Why use liposomes in drug delivery? <ul><li>Inactive: Unmodified liposomes gather in specific tissue </li></ul><ul><ul><li>Active: alter liposome surface with ligand (antibodies, </li></ul></ul><ul><ul><ul><li>enzymes, protein A, sugars) </li></ul></ul></ul><ul><ul><ul><li>Directly to site </li></ul></ul></ul>Physical: temperature or pH sensitive liposomes Drug targeting
Protection Decrease harmful side effects Pharmokinetics - efficacy and toxicity Changes the absorbance and biodistribution Change where drug accumulates in the body Protects drug Deliver drug in desired form Multidrug resistance Why use liposomes in drug delivery?
Release Affect the time in which the drug is released Prolong time -increase duration of action and decrease administration Dependent on drug and liposome properties Liposome composition, pH and osmotic gradient, and environment Why use liposomes in drug delivery?
Liposomes help improve: Therapeutic index Rapid metabolism Unfavorable pharmokinetics Low solubility Lack of stability Irritation
Current liposomal drug preparations: Type of Agents Examples Anticancer Drugs Anti bacterial Antiviral DNA material Enzymes Radionuclide Fungicides Vaccines Malaria merozoite, Malaria sporozoite Hepatitis B antigen, Rabies virus glycoprotein Amphotericin B In-111*, Tc-99m Hexosaminidase A Glucocerebrosidase, Peroxidase Duanorubicin, Doxorubicin, Epirubicin Methotrexate, Cisplatin*, Cytarabin Triclosan, Clindamycin hydrochloride, Ampicillin, peperacillin, rifamicin AZT cDNA - CFTR
No decrease in effectiveness of drug against fungi Liposomal formulation of AmB: Decrease in toxicity Exact Mechanism of liposomes not understood Cholesterol - only few %moles Phospholipid: AmB ratio Diffusion Lipid transfer AmB Lipid
Problems with liposomal preparations of drugs: Cost <ul><ul><li>Fungizone $40.58 Amphotec $2334 </li></ul></ul><ul><ul><li>Doxil $1200 per treatment, twice the cost of normal protocol </li></ul></ul><ul><ul><li>of chemotherapy and drugs </li></ul></ul>Lack long term stability (short shelf life) Freeze dry and pH adjustment Low “Pay Load” - poor encapsulation Physical and chemical instability Polar drugs and drugs without opposite charge Modifications
Possibility of new side effects Problems continued Efficacy
Studies with insulin show that liposomes may be an effective way to package proteins and peptides for use Clinical Trials for several liposomal formulations More studies on the manipulation of liposomes Future
References Journals Allen, Theresa M. "Liposomal Drug Formulations: Rationale for Development and What We Can Expect for the Future." Drugs 56: 747-756, 1998. Allen, Theresa M. "Long-circulating (sterically stabilized) liposomes for targeted drug delivery ." TiPs 15: 214-219, 1994. Allen, Theresa M. "Opportunities in Drug Delivery." Drugs 54 Suppl. 4: 8-14, 1997 Janknegt, Robert. "Liposomal and Lipid Formulations of Amphotericin B." Clinical Pharmacokinetics. 23(4): 279-291, 1992. Kim, Anna et al. "Pharmacodynamics of insulin in polyethylene glycol-coated liposomes." International Journal of Pharmaceutics. 180: 75-81, 1999. Quilitz, Rod. "Oncology Pharmacotherapy: The Use of Lipid Formulations of Amphotericin B in Cancer Patients." Cancer Control. 5:439-449, 1998. Ranade, Vasant V. "Drug Delivery Systems: Site-Specific Drug Delivery Using Liposomes as Carriers." Pharmacology. 29: 685-694, 1989. Storm, Gert and Daan J.A. Crommelin. "Liposomes:quo vadi?" PSTT 1: 19-31, 1998. Taylor, KMG and JM Newton. "Liposomes as a vecicle for drug delivery." British Journal of Hospital Medicine. 51: 55-59, 1994
Websites James, John S. "Doxil Approved for KS." www.immunet. org.imminet/atn.nsf/page/a-235-03. Wasan, Ellen. "Targeted Gene Transfer." Member.tripod.com/~rrishna/lipos1.html "Introduction to Controlled Drug Delivery Systems." www5.bae.ncsu.edu/bae/reearch/blak… k/otherprojects/drugDeliver_97/ http://www. Mssm.edu/medicine/thrombosis/phosphol.html "Doxorubicin." http://tirgan.com/adria.htm "Clinical Pharmacology Online." http://www.cponline.gsm.com/scripts/fullmono/showmono. "Drugstore.com" http://www.drugstore.com/pharmacy/prices/Amphotec. "Sequus' Doxil Becomes First Liposome Product Approved In U.S." www.slip.net/~mcdavis/ database/doxor_1 "Liposomes." www.collabo.com/liposom0.htm Paustin, Timothy. “Cellular Membranes.”www.bact.wisc.edu/microtextbook/bacterialstructure/Membranegen.html www.cbc.umn.edu/~mwd/cell_www/chapter2/membrane.html#PHOSPHOLIPIDS Books Jones, Macolm N. and Chapman, David. Micelles, Monolayers and Biomembranes . Wiley-Liss. New York (1995). Garrett, R. and Grisham C. Biochemistry , 2 nd ed. Saunders Colleges Publishing. New York (1999). 264.