LIPOSOMES.pptx

Liposomes
Preparation
Evaluation
Application

1. Guru Jambheshwar University of Science and  Techonology TOPIC NAME: LIPOSOMES. SUBMITTED TO: SUBMITTED BY: Dr.REKHA RAO KRISHAN NAGAR

2. LIST OF CONTENT: INTRODUCTION STRUCTURE OF LIPOSOMES COMPONENTS OF LIPOSOMES CLASSIFICATION OF LIPOSOMES CHARACTERIZATION OF LIPOSOMES TYPES OF LIPOSOMAL DRUG DELIVERY PLATFORMS REQUIREMENT MATERIAL FOR PREPARATION PREPARATION METHODS PHASE TRANSITION TEMPERATURE BIOLOGICAL CHANGES FACING LDDS EPR EFFECT ADVANTAGES & DISADVANTAGES APPLICATION OF LIPOSOMES

3. INTRODUCTION : A  liposome  is a small artificial  vesicle , spherical in shape, having at least one  lipid bilayer . Due to their hydrophobicity and/or hydrophilicity, biocompatibility, particle size and many other properties, liposomes can be used as  drug delivery  vehicles for  administration  of  pharmaceutical drugs  and  nutrients , such as  lipid nanoparticles  in  mRNA vaccines , and  DNA vaccines . Liposomes can be prepared by disrupting biological membranes (such as by  sonication ). Liposomes are most often composed of  phospholipids , especially  phosphatidylcholine and  cholesterol , but may also include other lipids, such as  egg ,  phosphatidyl ethanolamine , as long as they are compatible with  lipid bilayer structure. A liposome design may employ surface  ligands  for attaching to unhealthy tissue.

4. The major types of liposomes are the multilamellar vesicle (MLV, with several  lamellar phase   lipid bilayers ), the small  unilamellar liposome  vesicle (SUV, with one  lipid bilayer ), the large unilamellar vesicle (LUV), and the cochleate vesicle. A less desirable form are multivesicular liposomes in which one vesicle contains one or more smaller vesicles.

5. Structure Of Liposome:

6. STRUCTURAL COMPONENTS OF LIPOSOMES: The main components of liposomes are: Phospholipids Cholesterol Phospholipids : The phospholipids possess a hydrophobic tail structure and a hydrophilic head component.When dissolved in water, the hydrophobic tails mutually attract while the hydrophilic heads contact with the aqueous medium external and internal to the liposome surface. In this way, double lipid layers are formed which seal off to form small vesicles similar to the body cells and organelles. Natural phospholipids: PC-Phosphatidylcholine PE-Phosphatidylethanolamine PS-Phosphatidylserine

7. Synthetic phospholipids: DOPC-Dioleoylphosphatidylcholine DSPC-Disteroylphosphotidylcholine DOPE-Dioleoylhosphatidylethanolamine DSPE-DisteroyIphosphotidylethanolamine 2. Cholesterol: Cholesterol acts as a fluidity buffer, ie below the phase transition temperature, it makes the membrane less ordered and slightly more permeable; above the phase transition temperature it makes the membrane more ordered and stable .

8. CLASSIFICATION OF LIPOSOMES: Based on structural parameters: 1. MLV-Multilamellar vesicles(>0.5um) 2. OLV-Oligolamellar vesicles(0. 1-1 um) 3.UV Unilamellar vesicles(all size ranges) a. MUV –Medium Unilamellar Vesicles b. SUV-Small Unilamellar Vesicles(20-100nm) c. GUV-Giant Unilamellar Vesicles(>1 um) d . LUV-Large Unilamellar Vesicles(>100nm) 4. MVV/MV Multivesicular vesicles(>1 um)

9. Based on method of preparation: 1. REV- SUVs/OLVs made by reverse-phase evaporation method 2. MLV-REV-MLVs made by reverse-phase evaporation method 3. SPLV-›Stable plurilamellar vesicles 4. FATMLV-›Frozen and thawed MLV 5. VET-›Vesicles prepared by extrusion technique 6. DRV-De-hydrated rehydration method

10. Based upon composition and applications: 1. Conventional liposomes (CL) 2.  Fusogenic liposomes 3. pH sensitive liposomes 4. Cationic liposomes 5. Long circulatory(stealth) liposomes (LCL) 6.  Immuno-liposomes

11. CHARACTERIZATION OF LIPOSOMES: There are mainly three types of characterization of liposomes. They are: Physical characterization Biological characterization Chemical characterization

12. 1. Physical Characterization : S.No. Characterization parameters Analytical methods/instruments 1. Vesicle shape and surface morphology Transmission electron microscopy, Freeze-fracture electron microscopy 2. Surface charge Free-flow electrophoresis 3. Electrical surface potential and surface pH Zeta potential meastrements pH sensitive probes 4. Phase behavior Freeze_trachre electron microscopy Differential scanning colorimetery_ 5. Drug release Dittusion cell dia vsis

13. 2. BIOLOGICAL CHARACTERIZATION S.No. Characterization parameters Analytical method/Instrument 1. Sterility Aerobic or anaerobic cultures 2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test 3. Animal toxicity Monitoring survival rates, histology and pathology

14. 3. CHEMICAL CHARACTERIZATION S.No. Characterization parameter Analytical methods/instruments 1. Phospholipid concentration Barlett assay, stewart assay, TLC. 2. Cholesterol concentration Cholesterol oxidase assay, Ferric perchlorate method 3. Lysolecithin concentration Densitometer 4. Phospholipid peroxidation UV absorbance, lodometery , GLC. 5. Phospholipid hydrolysis & Cholesterol autooxidation HPLC & TLC 6. pH of liposomal dispersion pH meter 7. Osmolarity Osmometer

15. TYPES OF LIPOSOMAL DRUG DELIVERY PLATFORMS : In general, there are four key types of liposomal delivery systems : conventional liposomes sterically-stabilized liposomes ligand-targeted liposomes a combination of the above

16. 1. Conventional liposomes Conventional liposomes were the first generation of liposomes to be developed. They consist of a lipid bilayer that can be composed of cationic, anionic, or neutral ( phospho )lipids and cholesterol, which encloses an aqueous volume . Conventional liposomal formulations reduced the toxicity of compounds in vivo, through modifying pharmacokinetics and biodistribution to enhance drug delivery to diseased tissue in comparison to free drug. However, the delivery system was prone to rapid elimination from the bloodstream, therefore limiting its therapeutic efficacy . This rapid clearance was due to opsonization of plasma components and uptake by fixed macrophages of the reticuloendothelial system (RES), mainly in the liver and spleen .

17. 2. sterically-stabilized liposomes To improve liposome stability and enhance their circulation times in the blood, sterically-stabilized liposomes were introduced. The hydrophilic polymer, polyethylene glycol (PEG), has been shown to be the optimal choice for obtaining sterically-stabilized liposomes . The establishment of a steric barrier improves the efficacy of encapsulated agents by reducing in vivo opsonization with serum components, and the rapid recognition and uptake by the RES. This not only reduces the elimination of drugs by prolonging blood circulation and providing accumulation at pathological sites, but also attenuates side effects.

18. 3. Ligand-targeted liposomes Ligand-targeted liposomes offer a vast potential for site- specific delivery of drugs to designated cell types or organs in vivo, which selectively express or over-express specific ligands (e.g., receptors or cell adhesion molecules) at the site of disease . Many types of ligands are available, such as antibodies, peptides/proteins and carbohydrates. The coupling of antibodies, particularly monoclonal antibodies, to create immunoliposomes represents one of the more versatile ligands that can be affixed to liposome surfaces. One of the advantages of using monoclonal antibodies is their stability and higher binding avidity because of the presence of two binding sites on the molecule. Since lipid assemblies are usually dynamic structures, surface-coupled ligands have a high motional freedom to position themselves for optimal substrate-interactions.

19. 4. Combination of above The limited in vivo performance of immunoliposomes , due to poor pharmacokinetics and immunogenicity, has been a major hurdle to achieving their potential as effective site- specific drug carriers . Therefore, newer generation of liposomes have utilized a combination of the above design platforms to further improve liposomal targeting and associated drug delivery. For example, integrating target-specific binding of immunoliposomes with the steric stabilization of PEG (thereby creating long- circulating immunoliposomes ) has significantly improved the pharmacokinetics of immunoliposomes . Overall as a drug delivery platform, liposomes offer a dynamic and adaptable technology for enhancing the systemic efficacy of therapeutics in various diseases.

20. Requirement materials for preparation : Phospholipid like lecithin and all other kinds. Distilled water. Cholesterol. Tricine buffer . Methanol. Acetone. Chloroform. Diethyl ether. Ethanol.

21. Preparation methods : Due to phospholipids amphiphilic properties, they form accumulated complexes and protect their hydrophobic head groups from the aqueous phase while hydrophilic head groups stay in contact with water molecules, when they are located in aqueous environment. Sonication : Sonication is may be the most widely applied method for the preparation of liposomes and nanoliposomes . Sonication is a simple technique for dropping the size of liposomes and production of nanoliposomes . There are some disadvantages for this method such as very low internal volume/encapsulation efficiency, removal of large molecules and metal pollution from probe tip.

22. There are two major sonication techniques: Probe sonication Bath sonication . 1. Probe sonication : This is a common laboratory technique includes handling hydrated vesicles for several minutes with a titanium-tipped probe sonicator as explained in the following section. Dissolve an appropriate mixture of the phospholipid components, with cholesterol in chloroform with ratio of 2:1 v/v. Filter the mixture to eliminate slight insoluble ingre - dients then use a rotary evaporator to remove the sol- vents at temperatures above Tc or both lyophilization and spray drying can be used instead

23. Use a vacuum pump to remove traces of the organic solvents at pressures under 0.1 Pa. Add small quantity of glass beads with 550lm diameter to the flask comprising the dried lipids following by the addition of an appropriate aqueous phase such as distilled water or buffer. The aqueous phase can include chelating agents, stabilizers, salts, and the drug to be encapsulated . Disperse the dried lipids into hydration fluid by vortex mixing for 6 min so at this step micrometric MLV type liposomes are created. Use a probe sonicator and insert the tip of the sonicator in the MLV flask and run the sonicator . To avoid over-heating use 20 s ON, 20 s OFF intervals for a total period of 10–15min at this step nanoliposomes are obtained predominantly in the form of small unila - mellar vesicles (SUV).

24. 2. Bath sonication : In this method, the five first steps are as same as the probe sonicator method. In the following steps: Stow the bath sonicator with water (at room temperature) mixed with a couple of drops of liquid detergent and suspends the MLV flask in the bath sonicator and sonicate for a 30–45 min After preparation of product either in probe sonication or bath sonication method for annealing process put the final product at temperatures above Tc under inert circumstances such as nitrogen or argon for 1 h. Centrifuge could be applied to remove residual large particles to gain a clear suspension of nanoliposomes . There are important factors which influence the mean size and poly-dispersity index of vesicles such as temperature, sonication time, sample volume, sonicator tuning, and lipid composition and concentration.

25. Heating method : Most of the preparation methods of liposomes include the exploitation of potentially toxic solvents such as diethyl ether, methanol, and chloroform . The final product may contain remnants of these toxic solvents chip in potential toxicity and affect the stability of the lipid vesicles. Furthermore, to ensure the clinical appropriateness of the nanoliposomal products, the level of these solvents in the final formulations should be assessed and to decrease the time and cost of production particularly at the industrial scales, it would be much desirable to evade exploitation of these sol- vents in liposome preparation. Also, in preparation methods like microfluidization , the use of high pressures or high shear forces during liposome synthesis, cause harmful effects on the structure of the product. So, to overcome these kinds of obstacles, it would be better to apply substitute preparation methods like heating method which does not use potentially toxic solvents .

26. This method is explained in the following steps: Provide a proper mixture of the phospholipid ingredients, with proper amount of cholesterol in an aqueous phase under an inert condition such as nitrogen or argon for 1.5–2 h. Blend the lipid dispersions with the material (e.g. as drug) to be encapsulated and step up glycerol to a final volume concentration of 4%, then pour the mixture in the a heat-resistant bottle with six baffles. Put the bottle on a hot-plate magnetic stirrer at 900–1100rpm and a temperature above Tc and mix the sample for 35min. For cholesterol-containing formulations, it is b etter to dissolve cholesterol in the aqueous phase at lower temperature (110 C) in 1000 rpm for 20 min before adding the other ingredients. For annealing and stabilization process, place the finalproduct at temperatures above Tc under inert circum - stances such as nitrogen or argon for 1 h.

27. PHASE TRANSITION TEMPERATURE (Tc): At various temperatures, phospholipid membranes can exist in different phases. The transition from one phase to another can be detected by technique like micro-calorimetry. Factors like temperature, ionic strength, and pH are the important physicochemical properties of liposomes. Usually, these nano-structures have low permeability to the encapsulated material, but their permeability, might be changed at high temperatures. Phospholipids have an important thermal characteristic role in liposomes and they can undertake a phase transition (Tc) at temperatures lower than their concluding melting point .

28. At phase transition temperature, the lipidic bilayer drops much of its well-ordered packing while its fluidity enhances and this temperature depends on some factors like acyl chain length, Degree of saturation of the hydrocarbon chains, polar head group and ionic strength of the suspension medium. By declining chain length, presence of branched chains and bulky head groups and unsaturation of the acyl chains Tc falls. Important proper- ties such as accumulation, capability to deformation, fusion and permeability all of which can impact on the stability of liposomes depend on the phase behavior of liposomes.

29. BIOLOGICAL CHALLENGES FACING LIPOSOMAL DRUG DELIVERY SYSTEMS As with any foreign particle that enters the body, liposomes encounter multiple defense systems aimed at recognition, neutralization, and elimination of invading substances. These defenses include the RES, opsonization, and immunogenicity . While these obstacles must be circumvented for optimal liposome function, other factors such as the enhanced permeability and retention (EPR) effect can be exploited to enhance drug delivery.

30. The Reticuloendothelial System (RES) andLiposome Clearance The RES is the main site of liposome accumulation following their systemic administration . Primary organs associated with the RES include the liver, spleen, kidney, lungs, bone marrow, and lymph nodes. The liver exhibits the largest capacity for liposomal uptake followed by the spleen, which can accumulate liposomes up to 10-fold higher than other RES organs. The ability of the RES to sequester liposomes from the circulation is attributed to fenestrations in their microvasculature . Pore diameters in these capillaries can range from 100 to 800nm, which is large enough for the extravasation and subsequent removal of most drug-loaded liposomes (50–1000nm in size) . Liposomes are cleared in the RES by resident macrophages via direct interactions with the phagocytic cells. Uptake of liposomes by the RES is typically secondary to vesicle opsonization —that is, the adsorption of plasma proteins such as immunoglobulin, fibronectin , lipoproteins, and/or complement proteins onto the phospholipid membrane. However, in vitro studies have demonstrated that liposomal clearance via macrophages can also occur in the absence of plasma proteins.

31. The Enhanced Permeability and Retention(EPR) Effect Liposomes that have evaded both the RES and opsonization are subjected to the EPR effect . The EPR effect refers to the increased permeability of the vasculature that supplies pathological tissues (e.g., tumors and conditions involving inflammation). At these sites, deregulations in angiogenesis and/or the increased expression and activation of vascular permeability factors predominates, which leads to fenestrations that can range from 300 to 4700nm. This allows liposomes to extravasate and accumulate by passive targeting. For example, inflammation results in a dramatic change in blood vessel permeability as the capillary vasculature undergoes structural remodeling to allow leukocyte diapedesis into the peripheral tissue.

32. The width of the tight junctional regions between endothelial cells in vivo has been reported to range from 12 to 20nm , however exposure to inflammatory mediators increases permeability of the microvasculature , with the formation of gaps of up to 1 μ m. Pore sizes ranging from 0.2 to 1.2 μ m have been observed, though the size and number of pores are dependent upon the microenvironment of the pathological site. Importantly, all types of liposomal delivery systems are subjected to the EPR effect, with PEGylated liposomes having an advantage due to having reduced RES clearance and extended circulation time .

33. ADVANTAGES OF LIPOSOME: Provides selective passive targeting to tumour tissues (liposome doxorubicin). Increased efficacy and therapeutic index. Increased stability via-encapsulation Reduction in toxicity of the encapsulated agent. Site avoidance eftect . Improved pharmacokinetic effects (reduced elimination, increased circulation life times). Flexibility to couple with site-specific ligands to achieve active targeting.

34. DISADVANTAGES OF LIPOSOME: Production cost is high. Leakage and fusion of encapsulated drug/molecules. Sometimes phospholipids undergo oxidation and hydrolysis like reaction. Short half-life Low solubility.

35. APPLICATION OF LIPOSOME: Liposome as drug delivery vehicle Liposome as vaccine carrier Liposome in tumour therapy Liposome in gene delivery. Liposome as artificial blood surrogates Liposome as radio-pharmaceutical & radio-diagnostic carrier Liposome in cosmetics and dermatology Liposome in enzyme immobilization .

36. REFERENCES: https://en.wikipedia.org/wiki/Liposome . https:// www.ncbi.nlm.nih.gov / pmc /articles/PMC3599573/ . https:// www.slideshare.net / ArshadKhan63 /liposomes-119175712 . https:// www.slideshare.net / bharathpharmacist /liposomes-39685994 . Advances and Challenges of Liposome Assisted Drug Delivery written by Lisa Sercombe , Tejaswi Veerati , Fatemeh Moheimani , Sherry Y. Wu , AnilK.Sood andSusanHua . Recent advances on liposomal nanoparticles: synthesis, characterization and biomedical applications written by Yunes Panahi , Masoud Farshbaf , Majid Mohammadhosseini , Mozhdeh Mirahadi , Rovshan Khalilov , Siamak Saghfi & Abolfazl Akbarzadeh .

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