FORMULATIONFORMULATION OF OF ACETYL SALICYLIC ACIDACETYL SALICYLIC ACID ENCAPSULATED LIPOSOMES ENCAPSULATED LIPOSOMES S.S.W.B.M.G.U. EKANAYAKE, A.D.L.C. PERERA and D.N. KARUNARATNE. Department of Chemistry, University of Peradeniya. Drug encapsulationDrug encapsulation What is Encapsulation and What can it be used for? Inclusion of a substance inside a capsule mainly to control the release of the substance to be delivered. Encapsulation also helps to improve solubility and bioavailability of drugs.  ‘Controlled release’ strategies are highly prized in medicine since they can allow drugs to be absorbed more slowly, at a specific location in the body or at the say-so of an external trigger. Examples of nano and microcapsule Examples of nano and microcapsule designsdesigns for selected release mechanisms: for selected release mechanisms: Slow release - release payload slowly over a longer period of time Quick-release – break open upon contact with a surface (e.g. when pesticide hits a leaf ) Specific release - breaks open when a molecular receptor binds to a specific chemical (e.g., upon encountering a tumour or protein in the body) Moisture release - release contents in the presence of water (e.g. in soil) Heat-release – release on warming above a certain temperature pH release - nanocapsule breaks up only in specific acid or alkaline environment (e.g., in the stomach or inside a cell) Ultrasound release - the capsule is ruptured by an external ultrasound frequency Magnetic release - a magnetic particle in the capsule ruptures the shell when exposed to a magnetic field DNA nanocapsule – release foreign DNA into cells to express specific proteins (used for DNA vaccines) Matrices for drug Matrices for drug encapsulationencapsulation Polymer matrices for encapsulation Polymer-drug Polymer- drug-targeting conjugate ligand conjugate Dendrimer Polymeric NanoParticle with attached ligands Polymer micelle Matrices for drug Matrices for drug encapsulationencapsulation Lipid matrices for encapsulation Hydrophobic Hydrophilic Micelle Liposome Solid Lipid Nanoparticle Nanostructured lipid http://upload.wikimedia.org/wikipedia/commons/4/4d/Micelle_scheme-en.svg Why use liposomes in drug Why use liposomes in drug delivery?delivery? Reformulation of drugs in liposomes has provided an opportunity to enhance the therapeutic indices (TI) of various agents mainly through alteration in their bio distribution. The therapeutic applications of liposomes 1.Formulation aid Liposomes are made up of lipids which are relatively non-toxic, non-immunogenic, biocompatible and biodegradable molecules, and can encapsulate a broad range of water-insoluble (lipophilic) drugs 2.Site-avoidance delivery Liposomes are taken up poorly by tissues such as heart, kidney, and GI tract, which are major sites for toxic side-effects of a variety of anti neoplastic drugs. 3. Site-specific delivery Delivery larger fraction of drug to the target site and therefore, reducing exposure to normal tissues 4. Release Prolong time -increase duration of action and decrease administration OBJECTIVESOBJECTIVES Synthesize nanoparticulate liposomes using liquid crystals and egg yolk lecithin Encapsulate partially water soluble drug- Acetyl Salicylic Acid Study drug release for possible slow release/pH release Acetylsalicylic Acid (Aspirin)Acetylsalicylic Acid (Aspirin) Formula :CH3COOC6H4COOH Molecular wt. :180.16 Toxicity Oral rat :LD50: 200 mg/kg Physical properties : White, crystalline, weakly acidic substance, melting point 137°C, partially water soluble Uses • As a relief of headache and muscle and joint aches. • Reducing fever, inflammation, and swelling and thus has been used for treatment of rheumatoid arthritis, rheumatic fever, and mild infection. Encapsulation in liposomesEncapsulation in liposomes Thin Film Hydration Technique Entrap agents which are virtually insoluble in water and can be incorporated into the lipid bilayer during vesicle formation. These agents are generally treated as lipids and are mixed homogeneously with the lipid component prior to vesicle hydration step Preparation of LiposomesPreparation of Liposomes Egg yolk is a good source of phospholipids (PL) The main components of egg yolk Lecithin are Phosphatidylcholine (PC,80.5%, true lecithin from the chemical point of view) and Phosphatidylethanolamine (PE,11.7%) The carbohydrate liquid crystal used was β- sitosteryl 2,3,4,6,-tetra-O-acetyl-β-D- glucopyranoside Carbohydrate liquid crystals for Carbohydrate liquid crystals for preparation of liposomespreparation of liposomes Introduction ◦ Liquid crystal is the fourth state of matter that has properties of both the liquid and solid states ◦ A liquid crystal may flow like a liquid, but has the molecules in a liquid arranged and/or oriented in a crystal like way Carbohydrate Liquid CrystalsCarbohydrate Liquid Crystals ◦ Carbohydrate liquid crystals are usually composed of monosaccharide derivatives with one or more alkyl chains linked via (thio)-ether, ester, or amide linkages. ◦ Amphiphilic carbohydrates have been used as tools for molecular recognition in organized systems. Hydrophilic head (polar) Lipophilic tail (non polar) Figure : Structure of an amphiphilie Sugar hydrocarbon SYNTHESIS OF SYNTHESIS OF GLYCOLIPIDSGLYCOLIPIDS β-sitosteryl 2,3,4,6,-tetra-O-acetyl-β-D- glucopyranoside O H AcO H AcO H H OAcH OAc OAc CH3 CH3 HO CH3CH3 HCH3 H3C H H O H AcO H AcO H H OAcH O OAc CH3 CH3 H H CH3 CH3 CH3 H CH3 GLYCOSIDE BOND General principle: Glycoside bond formation reaction between aglycone and an activated anomeric center BF3.Et2O/CH2Cl2 Characterization of Characterization of β-sitosteryl 2,3,4,6,-tetra-O-acetyl-β-D-β-sitosteryl 2,3,4,6,-tetra-O-acetyl-β-D- glucopyranosideglucopyranoside 500 1000 1500 2000 2500 3000 3500 4000 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 wave number/cm -1 C O C C FT-IR Spectrum of β-sitosteryl 2,3,4,6-tetra-O- acetylglucopyranoside O H AcO H AcO H H OAcH O OAc CH3 CH3 H H CH3 CH3 CH3 H CH3 NMR Spectroscopic assignments:NMR Spectroscopic assignments: 1H-NMR Specrtum of β-Sitosteryl 2,3,4,6-tetra-O-acetylglucopyranoside 13C-NMR Spectrum of β- Sitosteryl 2,3,4,6-tetra-O- acetylglucopyranoside 2 1 12 13 4 3 14 15 6 5 7 8 17 16 11 10 9 21 27 22 23 24 25 26 29 28 19 20 HB7 9HD 6HA OHF8 10HC O O 3 O 14HMO2 O HK13 O4HL15 11HEG O 5 O 16HJ Lipid Part (Aglycon) Sugar Part O H AcO H AcO H H OAcH O OAc CH3 CH3 H H CH3 CH3 CH3 H CH3 POLARIZABLE MICROSCOPYPOLARIZABLE MICROSCOPY EXTINCTION BRUSHES According to the literature, The texture observed was characteristic for the hexagonal columnar phase due to the presence of extinction brushes. •Thermotropic liquid crystalline properties were shown by β- Sitosteryl 2,3,4,6-tetra-O-acetyl glucopyranoside. O H AcO H AcO H H OAcH O OAc CH3 CH3 H H CH3 CH3 CH3 H CH3 The cylinders are arranged in the best way of packing which is hexagonal lattice. A cylindrical arrangement with sugars surrounded by alkyl/steroidal groups. Preparation of Acetyl salicylic Preparation of Acetyl salicylic acid Encapsulated Liposomesacid Encapsulated Liposomes Compositions taken for the preparation of encapsulated liposomes Sample No Amount of PL/mg Amount of LC/ mg Total amount /mg Amount of Aspirin/mg 1 10.0 - 10 1.5 2 7.5 2.5 10 1.5 3 5.0 5.0 10 1.5 4 2.5 7.5 10 1.5 5 - 10.0 10 1.5 Encapsulation efficiencyEncapsulation efficiency Procedure: The suspensions centrifuged at 10000 rpm for 20 min at 4 oC The supernatants were collected quantitatively and filtered through 0.45μm membrane filters Measured UV- Absorbance of supernatants (275-280 nm) Encapsulation Efficiency % = Total aspirin-Free aspirin (in supernatant) X 100 Total aspirin Encapsulation efficiency Sample No Absorbance (supernatant) Concentration (supernatant) /ppm Encapsulation efficiency % 1 0.287 41.2 72.5 2 0.222 31.8 78.8 3 0.174 24.9 83.4 4 0.205 29.4 80.4 5 0.256 36.7 75.5 Absorbance of total aspirin (150 ppm) added = 1.046 Drug releaseDrug release Release of aspirin from liposomes (sample 3) was conducted by dialysis in a dialysis sac with 50.00 ml of deionised water and buffer solutions as the dialyzing medium 0 20 40 60 80 100 120 140 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 0.060 0.065 0.070 pH 2.0 pH 8.6 pH 7.0 A bs or ba nc e Time / min ConclusionConclusion Liposomes with 50% PL and 50% LC. (sample 3) has the highest encapsulation efficiency. Rapid drug release (burst release) was obtained at pH 8.6 and slow release observed at pH 2.0. This system would be able to prevent stomach irritation and unload its active ingredients in the intestine where the drug could be taken up. ReferencesReferences  Arcadio Chonn, Pieter R Cullis, Recent advances in liposomal drug delivery systems, Current Opinion in Biotechnology 1995, 6:698-708.  Paraveen Goyali, Kumud Goyali,Sengodan Gurusami, Vijaya Kumar , Ajit Singh,Omprakash Katare, Liposomal drug delivery systems – Clinical applications, Acta Pharm. 55 (2005) 1–25.  Liangfang Zhang ,Steve Granick, How to Stabilize Phospholipid Liposomes (Using Nanoparticles), Materials Research Laboratory and Department of Chemical & Biomolecular Engineering, University of Illinois, Urbana, Illinois 61801.  Luz E. Palacios , Tong Wang, Egg yolk lecithin fractionation and characterization, Department of Food Science and Human Nutrition Center for Crops Utilization Research,State University, Iowa, 50011-1061. AcknowledgementAcknowledgement Non specific funding from NSF, NRC and University of Peradeniya. Where nature finishes producing its shapes, there man begins, with natural things and with the help of nature itself, to create infinite varieties of shapes. Leonardo Da Vinci FORMULATION OF ACETYL SALICYLIC ACID ENCAPSULATED LIPOSOMES Drug encapsulation Examples of nano and microcapsule designs for selected release mechanisms: Matrices for drug encapsulation Slide 5 Why use liposomes in drug delivery? PowerPoint Presentation Objectives Acetylsalicylic Acid (Aspirin) Encapsulation in liposomes Preparation of Liposomes Carbohydrate liquid crystals for preparation of liposomes Carbohydrate Liquid Crystals SYNTHESIS OF GLYCOLIPIDS Characterization of β-sitosteryl 2,3,4,6,-tetra-O-acetyl-β-D-glucopyranoside NMR Spectroscopic assignments: POLARIZABLE MICROSCOPY Slide 18 Preparation of Acetyl salicylic acid Encapsulated Liposomes Encapsulation efficiency Slide 21 Drug release Conclusion References Acknowledgement THANK YOU