LIPOSOME CHATAP

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LIPOSOME

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content Introduction Mechanism of liposome formation Structural components of liposome's Advantages Disadvantages Classification of liposomes Methods of preparation of liposomes Characterization of liposomes Applications of liposomes Conclusion References

Liposomes (= vesicles):

3 Liposomes are concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. Liposomes were first produced in England in 1961 by Alec D. Bangham , who was studying phospholipids and blood clotting. The size of a liposome ranges from some 20 nm up to several micrometers. Liposomes (= vesicles)

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The lipid moecules are ususally phospholipids- amphipathic moieties with a hydrophilic head group and two hydrophobic tails. On addition of excess water, such lipidic moieties spontaneously originate to give the most thermodynamically stable conformation. In which polor head groups face outwords into the aqueous medium, and the lipidic chains turns inwords to avoid the water phase, giving rise to double layer or bilayer lamellar stractures.

Structural components of liposome's:

Structural components of liposome's Phospholipids Cholesterol

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Phospholipids Phosphatidylcholine . Amphipathic molecule Hydrophobic tail- 2 fatty acid chain containing 10-24 carbon atoms and 0-6 double bond in each chain Hydrophilic polar head- Phosporic acid bound to water soluble molecule Self organise in ordered supramolecular stracture when confronted (meet face to face) with solvent

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Some other conventional used phospholipids Naturally occurring phospholipids - PC: phosphatidylcholine - PE: Phosphatidylethanolamine - PS: Phosphatidylserine Synthetic phospholipids - DOPC: Dioleoylphosphatidylcholine - DSPC: Ditsearoylphosphatidylcholine - DOPE: Dioleoylphosphatidylethanolamine - DSPE: Ditsearoylphosphatidylethanolamine Polar Head Groups Three carbon glycerol Phospholipids in bilayer

Role of cholesterol in bilayer formation:

Role of cholesterol in bilayer formation Cholesterol by itself does not form bilayer structure. Cholesterol act as fluidity buffer After intercalation with phospholipid molecules alter the freedom of motion of carbon molecules in the acyl chain Restricts the transformations of trans to gauche conformations Cholesterol incorporation increases the separation between choline head group & eliminates normal electrostatic & hydrogen bonding interactions

ADVANTAGES OF LIPOSOMES:

ADVANTAGES OF LIPOSOMES VARIETY OF DRUGS GIVEN IN LOW DOSE AS ENCAPSULATED FOR STABILITY MINIMUM EFFECTIVE CONCENTRATION AND THEARAPEUTIC INDEX LOW TOXISITY DUE TO REDUSED EXPOSURE TO SENSITIVE TISSUES MINIMUM ADR/NO SIDE EFFECTS FOR ALTER KINETICS / DYNAMICS OF DRUGS CONTROL RELEASE BIOCOMPATIBLE/ BIODEGADABLE,NON TOXIC NONIMMUNOGANIC-SYSTEMIS/NONSYSTEMIC USE STABLE DELIVERTY FOE HYDROPHOBIC/PHILLIC AMPHIPHILIC DRUGS AS SUSTAIN REALEASE DEPOTS ENCAPSULATION POSSIBLE EX.Hb,INRTFRRONS,IL-2,ERYTHROPOATIN. POSSIBLE FOEMULATION-SUSPENTION,EMULSION,GEL, CREAM,LOTION, AEROSOL,RECONSTITUTED VESICLES

Disadvantages:

Disadvantages PHYSICAL/ CHEMICAL STABILITY VERY HIGH PRODUTION COST DRUG LEAKEGE/ ENTRAPMENT/ DRUG FUSION STERILIZATION SHORT BIOLOGICAL ACTIVITY / t ½ OXIDATION OF BILAYER …LIPIDS AND LOW SOLUBILITY RATE OF RELEASE and ALTERED BIODISTRIBUTION LOW THEARAPEUTIC INDEX and DOSE EFFECTIVENESS OVERCOMING RESISTANCE EXTENCIVE CLINICAL AND LABORATORY RESEARCH TO ACERTAIN LONG CERCULATING LIPOSOMES REPEATED IV ADMINISTRATION PROBLEMES

NEED OF Liposome in Drug Delivery :

NEED OF Liposome in Drug Delivery Pharmokinetics - efficacy and toxicity -Changes the absorbance and biodistribution -Deliver drug in desired form -Multidrug resistance Protection- -Decrease harmful side effects Change where drug accumulates in the body -Protects drug Release- -Affect the time in which the drug is released -Prolong time -increase duration of action and decrease administration -

Classification of liposomes:

Classification of liposomes Liposomes are classified on basis of - Structural parameters - Method of preparation - Composition and application

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Lamella : Types of vesicles on bases of lamella

Mechanism of liposome formation and subsequent processing to generate types of liposomes:

Mechanism of liposome formation and subsequent processing to generate types of liposomes

passive loading techniques :

passive loading techniques Mechanical Dispersion method Solvent Dispersion method Detergent Solubilisation method

Mechanical dispersion methods:

Mechanical dispersion methods

SOLVENT DISPERSION methods:

SOLVENT DISPERSION methods Note:- Organic solvent miscible with aqueous phase

DETERGENT SOLUBILISATIOIN methods:

DETERGENT SOLUBILISATIOIN methods Note:- Liposome size and shape depend on chemical nature of detergent, concentration and other lipid involved

active loading techniques :

Weak amphipathic bases accumulate in the aqueous phase of lipid vesicles in response to a difference in pH between the inside and outside of the liposomes ( pHin & pHout ) Two steps process generates this pH imbalance and active (remote) loading. Vesicles are prapared in low pH solution, thus generating low pH within the liposomal interiors, followed by addition of the base to extraliposomal medium. Basic compounds, carrying amino groups are relatively lipophipic at high pH and hydrophilic at low pH. In two chambered aqueous system separated by membrane liposomes , accumulation occurs at the low pH side, under dynamic equilibrium conditions. Thus the unprotonated form of basic drug can diffuse through the bilayer The exchange of external medium by gel chromatography with neutral solution Weak base doxorubicine , adriamycin and vincristine which co-exist in aqueous solutions in neutral and charged forms have been sucessfully loaded into preformed liposomes via the pH gradient method. active loading techniques AFTER DRYING IN PROCESS FILM/CAKE OF LIPID IS FROM STACKS OF LIPID BILAYER FORM SWELLING IN FLUID SHEET IS SELF CLOSE LOADING OF DRUG ON p H- GRADIENT TECHNIQUE FORMATION OF BILAYER (LIPOSOMES) IF DRUG

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LIPID FILM HYDRATION BY HAND SHAKING FREEZE DRYING OR NON HAND SHAKING MICROEMULSIFICATION SONICATION MEMBRANE EXTRUSON DRIED RECONSTITUTED VESICLES ETHANOL INJECTION ETHER INJECTION DOUBLE EMULSION REVERSEPHAS EVAPOURATION VESICLES STABLE PLURI LAMELLER VESICLES CHROMATIGRALPY DIFFUSION VESICLES LIKE…. RECONSTITUTED & SANDAI VIRUS ENVELOPE DETERGENT REMOVAL FORM MIXED MICELLES BY DIALYSIS

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Method Vesicles Mechanical methods Vortex or hand shaking of phospholipid dispersions MLV Extrusion through polycarbonate filters at low or medium pressure OLV, LUV Extrusion through a French press cell “ Microfluidizer ” technique Mainly SUV High-pressure homogenization Mainly SUV Ultrasonic irritation SUV of minimal size Bubbling of gas BSV Methods based on replacement of organic solvent(s) by aqueous media Removal of organic solvent(s) MLV, OLV, SUV Use of water-immiscible solvents: ether and petroleum MLV, OLV, LUV Ethanol injection method LUV Ether infusion (solvent vaporization) LUV, OLV, MLV Reverse-phase evaporation Methods based on detergent removal Gel exclusion chromatography SUV “Slow” dialysis LUV, OLV, MLV Fast dilution LUV, OLV Other related techniques MLV, OLV, LUV, SUV

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SOLVANT eg.CHCl 3 LIPID e.g.. LICITHIN DISPERSION FORMATIONN Encapsulated solute Separate liposome's from supernatatant by centrifugation, gel filtration/ sonication or dialysis or by addition of buffers + drug. FILM OF LIPID OCCURS AT SIDES OF VESSELS BASIC METHOD OF FORMULATION OF LIPOSOMES

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Gel Filtration Column Chromatography Purification of Liposomes

Lipid Hydration Method:

Lipid Hydration Method The mechanical energy required for swelling of lipids and dispersion of casted lipid film is imparted by manual agitation (hand shaking technique) The % encapsulation efficiency as high as 30% is achieved due to loss of water soluble component during swelling and entrapped only 10-15%. On other hand lipid soluble drug encapsulated 100% efficiency.

PROLIPOSOMES:

PROLIPOSOMES To increase the surface area of dried lipid film and to facilitate continuous hydration and lipid is dried over the finally divided particulate support i.e.- NaCl , Sorbitol , or other polysaccharides. These dried lipid coated particulates are called as proliposomes Proliposomes form dispersion of MLVs on addition of water, where support is rapidly dissolved and lipid film hydrate to form MLVs Methods overcome the stability problem and entrapment efficiency doesn’t matter when formation of stable liposome.

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Sonication Method PROBE SONICATOR : is employed for dispersions, which require high energy in a small volume (e.g., high concentration of lipids, or a viscous aqueous phase) Disadvantage - lipid degradation due to high energy and sonication tips release titanium particles into liposome dispersion BATH SONICATOR: The bath is more suitable for large volumes of diluted lipids. Method: Placing a test tube containing the dispersion in a bath sonicator and sonicating for 5-10min(1,00,000g) which yield a slightly hazy transparent solution. Using centrifugation to yield a clear SUV dispersion

French pressure cell liposomes:

French pressure cell liposomes This techniques yields rather “ uni or oligo lamellar liposomes ” of intermediate size of 30-80 nm in diameter depending on the applied pressure. Dispersion of MLVs can be converted to SUVs by passage through a small orifice under high pressure. MLV dispersion are placed in the French pressure cell and extruded at about 20,000psi at 45 0 C By multiple extrusion i.e.., 4.5 passed about 95% of MLVs get converted into SUVs which can be determined by size exclusion chromatography.

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Micro Emulsification Liposomes (MEL) The fluid collected can be recycled through the pump and interaction chamber until vesicles of the spherical dimension are obtained. After a single pass, the size of vesicles is reduced to a size 0.1 and 0.2um in diameter. “Micro Fluidizer” is used to prepare small MLVs from Concentrated lipid dispersion The lipids can introduced into fluidizers, either as a dispersion of large MLVs or as a slurry of unhydrated lipids in organic medium. Microfluidizer pumps the fluid at very high pressure(10,000psi, 600-700 bar) through a 5um orifice. Then it is forced along defined micro channels, which direct two streams of fluid to collide together at right angles at a very high velocity, thereby affecting an efficient transfer of energy.

Vesicles prepared by Extrusion TechNIQUES (VETs):

Vesicles prepared by Extrusion TechNIQUES (VETs) It is used to process LUVs as well as MLVs. Liposomes prepared by this tech. are called as membrane filter extrusion liposomes The 30% capture volume can be obtained using high lipid conc. The trapped volume in this process is 1-2 litre /mole of lipids It is due to their ease of production, readily selectable vesicle diameter, batch to batch reproducibility & freedom from solvent or surfactant contamination is possible

Freeze thaw sonication method (FTS):

Freeze thaw sonication method (FTS) The method is based on freezing of a unilamellar dispersion & then thawing at room temp for 15 min. Thus the process ruptures & refuses SUVs during which the solute equilibrates between inside & outside & liposomes themselves fuse & increase in size. Entrapment volume can be upto 30% of the total vol. of dispersion. Sucrose, divalent metal ions & high ionic strength salt solutions can not be entrapped efficiently

DRIED-RECONSTITUTED VESICLES:

DRIED-RECONSTITUTED VESICLES Liposomes obtained by this method are usually “ uni or oligo lamellar” of the order of 1.0um or less in diameter. SUVs in aqueous phase SUVs with solutes to be entrapped Freeze dried membrane Solutes in uni lamellar vesicles Solutes in uni or oligo lamellar vesicles FST method DRV method Rehydration Film stacks dispersion Aqueous phase Thawing Sonication (15-30 sec)

LIPOSOMES FROM PREFORMED VESICLES:

Vesicles prepared by using fusion of SUVs by fusogenic agents or by change in the microenvironment of the systems LIPOSOMES FROM PREFORMED VESICLES

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This method is used to transform MLVs to LUVs using a change in the pH of the dispersion thus avoiding the use of Sonication and high-pressure application. The process is an electrostatic event and termed as pH induced vesiculation The transient change in pH bring about an increase in the surface charge density of the lipid bilayer , which induces spontaneous vesiculation Preformed MLVs (Prepared using hand shaking, freeze thawing) pH 2.5-3.0 exposed to high pH ie.,11.0 less than 2min (1M NaOH ) pH reduced by 0.1M HCl until pH 7.5 SUVs dispersion pH Induced Vesiculation

CALCIUM INDUCED FUSION:

CALCIUM INDUCED FUSION Calcium induced fusion method is principally based upon the concept of aggregation and fusion of acidic phospholipid vesicles in the presence of calcium. SUVs formed from using sonication buffer ( NaCl 0.385g, histidine,31mg,Tris-base24.2mg,EDTA3.72mg, water100ml,pH7.4) as hydrating fluid. The large liposomes and lipid particles are removed by centrifugation at 100,000g. Equimolar proportion of Calcium solution(CaCl2) is added to Phospholipids in the supernatant, resulting in white precipitate, incubated for 60min at 37 ˙C and finally Ca-EDTA complex is removed by dialysing the dispersion overnight against a litre of phosphate saline buffer and separate pellets

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Cochleates are formed when SUVs made from negatively charged lipids mainely phosphotidylserin (PS) fuse into cylindrical rolls, termed as Cochleate cylinders. Small unilamellar vesicles Negatively charged lipids ( Phosphatidylserine PS) addition of Ca ++ Cylindrical rolls ( Cochleate cylinders) removal of Ca ++ by EDTA or ion exchange or Precipitation Large unilamellar vesicles COCHLEATE METHOD

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An ethanol solution of lipids is injected rapidly through a fine needle into an excess of saline or other aq. medium This method has low risk of degradation of sensitive lipids The vesicles of 100 nm size may be obtained by varying the conc. Of lipid in ethanol or by changing the rate of injection of ethanol solution in preheated aqu . solution. Limitation -solubility of lipids in ethanol & vol. of ethanol that can be introduced into medium(7.5%v/v max) Difficulty to remove residual ethanol from phospholipid membrane Involves mixing of organic phase into aqu . Phase at the temp. of vaporizing the organic solvent It has low encapsulation efficiency Ethanol injection :- Ether injection :-

Rapid solvent exchange vesicles (RSEVs):

Rapid solvent exchange vesicles (RSEVs) Lipid mixture is transferred between pure solvent & a pure aq.environment . Organic sol. of lipids through orifice of syringe under vacuum into a tube containing aqueous buffer. The tube is mounted on vortexer . It manifest high entrapment volumes

Reverse phase evaporation method:

Reverse phase evaporation method

Characterization of liposomes:

Characterization of liposomes PHYSICAL CHARECTIRISATION Vesicles size/shape/morphology Surface -charge/electrical potential Phase bahaviour / lamellarity Drug release % capture /free drug CHEMICA L CHARECTIRISATION Phospholipids /lipid concentration Drug concentration PH / Osmomolality Antioxidant degradation Phospholipids / cholesterols – peroxidation /oxidation/hydrolysis BIOLOGICAL CHARECTIRISATION Sterility Pyrogenisity Animal toxicity Plasma Stability:

Liposome characterization with their quality control assays:

Liposome characterization with their quality control assays Characterization parameters Analytical methods/instrumentation Chemical characterization Phospholipid conc. Cholesterol conc. Drug conc. Phospholipid peroxidation Osmolarity Barlet assays/Stewart assays, HPLC HPLC Monograph UV absorbance, iodometric & GLC Osmometer Physical characterization Vesicle shape & surface morphology Size & size distribution Submicron range Micron range TEM, Freeze fracture electron microscopy TEM TEM,FFEM, photon correlation spectroscopy, laser light scattering, gel permeation Biological characterization Sterility Pyrogenicity Animal toxicity Aerobic or anaerobic cultures LAL test Monitoring survival rates, histology & pathology

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Vesicle shape & lamellarity & Vesicle size & size distribution :- PHYSICAL CHARECTIRISATION Microscopic techniques :- Optical Mycroscopy :- D etermination of gross size distribution of large vesicles preparations such as MLVs & Morphological structure of liposome various tech. include light microscopy, fluorescent microscopy, electron microscopy, laser light scattering, field flow fractionation, gel permeation & gel exclusion, zetasizer

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Freez Fracture Electron Microscopy Negative Stain Electron Microscopy Transmission Electron Microscopy Scanning Electron Microscopy Cryo -Electron Microscopy Laser Light Scattering Tchnique Fluorescence Electron Microscopy Confocal Laser Light Scnning Microscopy Electron Microscopic Techniques :-

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Freez Fracture Electron Microscopy The freeze-fracture/freeze etch technique starts with rapid freezing of a cell. Then the frozen cells are cleaved along a fracture plane. This fracture plane is in between the leaflets of the lipid bilayer , The two fractured sections are then coated with heavy metal (etched) and a replica is made of their surfaces. This replica is then viewed in an electron microscope.

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Negative Stain Electron Microscopy Negative stain electron microscopy visualizes electron transparent liposomes as bright areas against a dark background. Negative stains used in the TEM analysis is ammonium molybdate

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Transmission Electron Microscopy Transmission electron microscopy ( TEM ) is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film , or to be detected by a sensor such as a CCD camera .

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Scanning Electron Microscopy A scanning electron microscope ( SEM ) is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography , composition, and other properties such as electrical conductivity .

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Cryo -Electron Microscopy Is form of transmission electron microscopy known as Cryo transmission electron microscopy ( cryo -TEM) where the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures). Cryo -TEM of liposome dispersion. Scale bar is 200 nm. CryoEM image of GroEL suspended in vitreous ice at 50,000X magnification

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Laser Light Scattering Tchnique

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The "fluorescence microscope" refers to any microscope that uses fluorescence to generate an image Fluorescence Electron Microscopy

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Confocal Laser Light Scnning Microscopy Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane . [1] It enables the reconstruction of three-dimensional structures from the obtained images . Technique for obtaining high- resolution optical images with depth selectivity i.e. Penetration and Permeation Studies

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Zetasizer Zeta potential is an important and useful indicator of particle surface charge, which can be used to predict and control the stability. In general, particles could be dispersed stably when the absolute value of zeta potential was above30mV due to the electric repulsion between particles

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Gel permeation :- Preferably used for the size distribution determination of liposomes Ultracentrifuge:- Used for size distribution of liposomes Encapsulation efficiency & trapped volume :- Determines % of the aq. Phase & hence % of water soluble drug which is entrapped & expressed as % entrapment/mg lipid Drug Content

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Trapped volume(0.5-30 µ l/mg) :- The internal or trapped volume is the aqueous entrapped volume per unit quantity of lipid & expressed as µ l/ µ mol or µ l/mg of total lipid. Radioactive markers are used to determine the internal volume. Vesicle fusion measurements :- It has been studied in case of cationic liposomes , PH sensitive liposomes . fusion has been monitored using a fluorescence resonance energy transfer (RET) between two lipid analogues originally placed in separate vesicle population that measures intermixing of membrane lipids

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Phase response & transitional behavior: - Lipid bilayers can exists in a low temperature solid ordered phase & above certain temp in a fluid disordered phase . Phase behavior of liposomal membrane determines prop. such as permeability, fusion, aggregation & protein binding Thermodynamic methods :-In differential scanning microcalorimeter , the heat required by liposomes to maintain a steady upward rise in temp is plotted as a function of temperature

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Elasticity Measurement of Liposomes Extrusion Method Liposomal formulations were extruded through filter membrane (pore diameter 50 nm), using a stainless steel filter holder having 25-mm diameter, by applying a pressure of 2.5 bar. The quantity of vesicle suspension, extruded in 5 minutes was measured. Skin Permeation Study Franz Diffusion Cell

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1.Phospholipid conc. is determined in terms of lipid phosphorus content using Barlet assay/Stewart assay or TLC 2.Cholesterol conc. is determined using Ferric perchlorate method/Cholesterol oxidase assay 3.Lysolecithin:-which is one of the major product of hydrolysis is estimated using densitometry 4.Phospholipid peroxidation is determined by UV absorbance, iodometry , GLC technique. 5.Phospholipid hydrolysis is determined using HPLC & TLC 6.Cholesterol auto oxidation can be determined by HPLC & TLC CHEMICA L CHARECTIRISATION

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Sterility:- Aerobic or anaerobic cultures Pyrogenisity :- LAL test Animal toxicity:- Monitoring survival rates, histology & pathology Plasma Stability :- Cytotoxicity Assay, HPLC Assay BIOLOGICAL CHARECTIRISATION

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Two most frequently encountered biological events that the administered liposomal system undergoes are phagocytosis or antigen presentation via the macrophages of the RES system Opsonins which are proteinaceous components of serum adsorb onto the surface of liposomes thus making these exogenous materials more palatable & conductive to phagocytes High density lipoprotein removes phospholipid molecules from bilayered vesicular systems The molecular origin of these interactions are mostly long range electrostatic, Vander waals & short range hydrophobic interactions of particulate surface with macromolecules in the serum Stability after systemic administration

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Liposome-cell interaction

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Vesicle-Skin Interaction Study by TEM and SEM From animals ultra thin sections were cut collected on formvar -coated grids and examined Fluorescence Microscopy

Stability of liposomes:

Stability of liposomes The stability in vitro which covers the stability aspects prior to the administration of the formulation & with regard to the stability of the constitutive lipids The stability in vivo which covers the stability aspects once the formulation is administered via various routes to the biological fluids. It includes stability aspects in blood if administered by systemic route or in gastrointestinal tract if administered by oral or peroral routes Stability in vitro :- method of formulation, nature of amphiphile & encapsulated drug, manipulate membrane fluidity/rigidity & permeability characteristics. Storage temp. of these dispersions must be defined & controlled Liposomal phospholipids can undergo degradation such as oxidation & hydrolysis Storing the vesicles at 4°C ± 0.5°C. Vesicle size, zeta potential, and entrapment efficiency of the vesicles was measured after 180 days using the method described earlier.

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1. Lipid oxidation & Peroxidation Lipid peroxidation measurement is based on disappearance of unsaturated fatty acids or appearance of conjugated dienes . It can be prevented by minimizing use of unsaturated lipids, use of oxygen, argon or nitrogen environment, use of antioxidant such as Alpha tocopherols or BHT or use of light resistant containers for storage of liposomal preparations 2. Lipid hydrolysis It leads to lysolecithin formation The inclusion of charged molecule in the bilayer shifts the electrophoretic mobility & makes it positive with addition of stearylamine or negative with dicetyl phosphate thus prevents liposomal fusion/swelling or aggregation 3.Long term & Accelerated stability High temp. testing(>25 0 C) is universally used for heterogenous products. Various laboratories store their products at temp ranging from 4 0 C to 50 0 C.

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72 STEROIDAL DRUG DELIVERY AS VESICLES /VACCINES GENES THEARAPY/ANTISENCE TECHANOLOGY VEHICLES FOR MACROMOLECULES AS CYTOKINES IMMUNOLIPOSOME THEARAPY RADIOPHARMACEUTICAL/REDIOCARREIR IN ENZYME IMMOBILYZATION/BIOREACTION INSULIN DRUG DELIVERY CHRONIC DISESES…………. ARTERIOSELEROSIS,CYSTIC,FIBRROSIS,HEMOPHILIA, SIKLECELL,ANEMIA,SRCOMA,CHRONIC INFLAMATION AND PAIN AS DRUG CARRIER APPLICATION OF LIPOSOMES

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LIPOSOMES THERAPY DRUGS /USE AIDS Azidotymidine Cancer Cisplatin,Taxol,Doxorubicin Malaria Primaquine , Chloroquine , Artemisinin Gramicidin A lung Isoniazid,Rifampicin,Budesonide Infectious Diseases e.g. skin Amphotericin,Antimony,Pentamidine DRUGS Antibiotics, Antifungal Disinfectant, Immunosuppressive agents Dermatology and Cosmetology Local anesthetic e.g. Lidocaine and Benzocaine,Gentamycin,Cefazolin immunological (Vaccine) Adjuvant Hepatitis A rabies virus, Measles virus, influenza virus Herpes virus, HIV-1 and Vesicular stomatitis DIEBETIS INSULIN / Hypoglysemics Radiodiagnostic Carriers γ- scintigraphy , Magnetic resonance (MR), Computer tomography (CT) and Ultrasonography (US) of tumors

Liposomes as drug delivery vehicles:

Liposomes as drug delivery vehicles Enhanced drug solubilization ( amphotericin B, minoxidil , paclitaxel , cyclosporin ) Protection of sensitive drug molecules (cytosine arabinose , DNA, RNA, ribozymes ) . Liposomes in antimicrobial, antifungal & antiviral therapy Intracellular pathogens ( protozoal , bacterial,& fungal) harbour in liver & speen & hence drugs can be targeted to these organs.e.g.Intracellular localization of pathogenes necessitates administration of high doses of cytotoxic drugs for effective killing of pathogen causing side effects Drug to be targeted to macrophages in such a way that interaction of free drug with nontarget tissues could be minimized pathogen Treatment with liposomal Amp B results in lower toxicity & increased survival times

Liposomes in tumour therapy:

Liposomes in tumour therapy Targeting strategies using liposomes are Natural targeting of conventional liposomes (passive vectorization ) Use of long circulatory (stealth liposomes ) Use of ligand mediated targeting (active targeting) The use of anti-receptor antibodies on the tumour vascular endothelium Use of stealth liposomes & ligands mediated targeting in combination Drug Target disease Status Product Doxorubicin Kaposi's sarcoma Approved SEQUUS Daunosome Breast cancer Approved NeXstar,USA Nystatin Systemic fungal infections Phase II Aronex, USA Amikacin Serious bacterial infections Phase II NeXstar,USA Vincristin Solid tumours Preclinical dev. NeXstar,USA

Liposomes in gene therapy:

Liposomes in gene therapy Recombinant DNA tech., studies of gene function & gene therapy all depend on delivery of nucleic acids( genetic material) into cells in vitro & in vivo. Gene can be viral (adenovirus, retrovirus) & non viral( liposomes & lipid based systems, polymers & peptides) Type of vectors Advantages Disadvantages Viral vectors (Adenovirus, retrovirus & adeno-associated virus) Relatively high transfection efficiency Immunogenicity, presence of contaminants & safety Vector restricted size limitation for recombinant gene Non viral vectors (liposomes/lipid based systems, polymers & peptides) Favorable, pharmaceutical issue-GMP, stability, cost Plasmid independent structure Low immunogenicity Opportunity for chemical/physical manipulation low transfection efficiency Advantages & disadvantages of viral & non viral systems

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P H sensitive liposomes The P H sensitive liposomes have been reported as plasmid expression vectors for the cytosolic delivery of DNA. P H sensitive immunoliposomes P H sensitive liposomes have been developed to release their contents in response to an acid machinery within endosomal system following receptor mediated endocytosis of the immunological targeting ligand Fusogenic liposomes & Virosomes They fuse & merge with cell membranes & directly introduce molecules (entrapped or anchored) into cytoplasm & avoiding route followed by conventional liposomes . Fusion can be mediated by PEG, glycerol & Polyvinyl alcohol or by reconstituted fusogenic viral membrane based liposomes are termed as Virosomes

Liposomes as immunological (vaccine) adjuvant:

Liposomes as immunological (vaccine) adjuvant A non immunogenic substance may be converted into immunogenic Hydrophobic antigens may be reconstituted Small amount of antigen may be suitable as immunogens Multiple antigens may be incorporated into the single liposomes Adjuvant may be incorporated with antigens into the liposomes Longer duration of functional antibody activity may be achieved Soluble synthetic antigen may be presented as membrane associated antigens in an insoluble liposomal matrix

Liposomal vaccines:

Liposomal vaccines New vaccines that are based on recombinant protein subunits & synthetic peptide antigens are usually non-immunogenic, hence need of immunopotentiation is realized Alum was used as vaccine adjuvant The first liposome based vaccine (against hepatitis A) that has been licensed for use in human is an IRIV vaccine which are spherical, unilamellar vesicles with a diameter of 150nm. IRIVs are prepared by detergent removal of influenza surface glycoproteins & a mixture of natural & synthetic phospholipids containing 70% egg yolk phosphatidylcholine,20 % synthetic PE & 10 % envelop phospholipids originating from H1N1 influenza virus

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The main purpose is to activate macrophages & render them tumouricidal . They acquire ability to recognize & destroy neoplastic cells both in vitro & in vivo. Liposomes in Immunodiagnosis 1. LILA assays (liposome immune lysis assay) has been implicated in the detection of serum components such as carcinoembryonic antigen,C -reactive protein & other serum protein which serve as diagnostic tools for cancer 2 . LILA sandwich method has been used to detect many important antigens in serum, which are useful indicators of various abnormalities Liposomes as a carrier of Immunomodulator

Liposomes in Dermatology and Cosmetology:

Liposomes in Dermatology and Cosmetology Similar to biological membrane they can navigate water soluble & lipophilic substances in different phases They mimic the lipid composition & structure of human skin, which enables them to penetrate the epidermal barrier Liposomes are biodegradable & nontoxic, thus avoiding local/systemic side or toxic effects Moisturizing & restoring action of constitutive lipids Liposomes may act as localized drug depots in skin resulting in sustained release of drug, thus improving therapeutic index of drug at target site while reducing toxicity profile to minimum

Liposomes as Radiopharmaceutical & radiodiagnostic carriers:

Liposomes as Radiopharmaceutical & radiodiagnostic carriers Liposomes loaded with contrast agents are suitable for contrast agents are substances which are able to absorb certain types of signal much stronger than surrounding tissue Radiodiagnostic application include liver & spleen imaging, tumour imaging, imaging cardiovascular pathologies, visualization of inflammation & infected sites, brain imaging, visualization of bone marrow The RES avoidance of contrast agents can be achieved by using targeted liposomes like immunoliposomes

Liposomes as Red cells substitutes & artificial RBCs:

Liposomes as Red cells substitutes & artificial RBCs Synthetic & semisynthetic blood substitutes includes recombinant haemoglibin , glutaraldehyde cross linked haemoglobin,haemoglobin encapsulated liposomes Liposome encapsulated haemoglobin products are being investigated as artificial RBCs Researchers reported completely synthetic amphiphilic heme derivative (lipid heme ) & incorporated them into the hydrophobic centre of the bilayer membrane of the phospholipid vesicles, which has excellent oxygen carrying & transporting abilities

REFERENCES:

REFERENCES Jain N.K., Controlled & Novel Drug Delivery, CBS Publications, New Delhi Jain N.K., Advances in Controlled & Novel Drug Delivery, CBS Publications, New Delhi. Vyas S.P. and Khar R.K., Controlled drug delivery- Concepts & Advances, Vallabh Prakashan , New Delhi. Vyas S.P. and Khar R.K., Targeted & Controlled drug delivery- Novel Career System, CBS Publications, New Delhi. Chien Y, Novel Drug Delivery System, Mercel Decker Publications. Lee & Robinson, Controlled Drug Delivery, Second Edition, Mercel Decker Publications. Swarbrick J and Boylon J.C., Encyclopedia of Pharmaceutical Technology, Vol. 1-3, Mercel Decker Inc. 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 (serially stabilized) liposome for targeted drug delivery ." Tips 15: 214-219, 1994. Vesicular drug delivery system by R.S.R. Murthy.

QUESTIONS ASK IN NMU UNIVERSITY EXAMINATION:

What are liposomes . Discuss in detail mechanical dispersion method of liposome preparation with diagrame . Give the mechanism of liposome formation. Discuss about solvent dispersion method of passive loading for liposomes . Give in nutshell mechanism of liposome formation QUESTIONS ASK IN NMU UNIVERSITY EXAMINATION

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