Liposomes

liposome

1. PREFORMULATIONOF LIPOSOMES And Microsphere Prepared by :- Guide :- Sneha A. Chavan Dr. (Mrs.) Aparna Palshetkar Department of Pharmaceutics M - Pharmacy 1 st Year

2. What is liposome and Microsphere

3. Preformulation Definition : It is a stage of formulation development where physical and chemical properties of drugs alone and when combined with excipients are studied. Objective : To generate information useful to formulator in developing stable and bioavailable dosage forms.

4. Bulk characterization

5. Solubility characterization

6. Stability characterization

7. Shape

8. LIPOSOME MICROSPHERE Size Ranges from 20nm – 3 µm ranges from 1-1000 µm Vesicle Shape or size Electron Microscopic Technique the average size of vesicle automated diffraction technology . Mean particle size, frequency distribution profile and polydispersibility index are analyzed output that can be interpreted for the developed microparticles. Optical microscopy determining particle size of microsphere larger than 10 µm. Imaging analysis facilities (Imaging and analysis software) useful in determining the size distribution of microsphere High resolution TEM size of particles by point to point resolution

9. Vesicle size and Distribution of liposome 1. Microscopic technique Optical microscopy Fluorescent microscopy ( if fluorescent probe is included in either lipid or aqueous domain ) Cryo –Transmission electron microscopy ( Cryo - TEM) Scanning Electron microscopy ( SEM ) Negative stain Transmission electron microscopy ( TEM ) Freeze fracture microscopy 2. Diffraction and scattering technique Laser light scattering Photon correlation spectroscopy 3.Hydrodynamic technique Field flow fractionation ( FFF ) Technique Gel permeation and gel exclusion and zetasizer ultracentrifuge zl

10. 2. Surface charge

11. Surface charge The charge of microparticles can be analyzed as it indicates the dispersion of the micron size particles in a medium. Surface charge decides the degree of aggregation upon storage or even reconstitution, which further leads to alteration in the stability, Syringeability of microsphere. Zeta potential is measured for microparticles and it indicates the electrostatic interaction. It is measured by micro-electrophoretic technique where the movement of particles is measured under the influence of known electric field. Alternatively electrophoretic light scattering technique can be used for measuring zeta potential of microsphere.

12. The surface charge can be calculated by estimating the mobility of the liposomal dispersion in a suitable buffer The lipid – cell interaction can be governed by the nature and density of charge on the liposome surface. Charging the lipid composition can alter the nature and charge on the liposome . T he interaction of neutrally charged liposome with the cell is almost negligible. But positively charged liposomes are cleared more rapidly after systemic administration. Unlike negatively charged liposomes, cationic liposomes deliver the contents to cells by fusion with cell membrane

13. Aqueous two phase partitioning system

14. 3. Inter Excipient And Drug Excipient Compatibility Drug excipient reaction might produce new impurity or degradant of undesired properties. Analytical methodology ( TLC, HPLC, GC, LC-MS, GC-MS, UV-VIS, FTIR, NMR) developed for quantitative and qualitative detection of Impurity species in the formulation. Employed for the characterization of the chemical instability of the excipient as well as API-excipient mixture. Drug excipient interactions leads to chemical alteration and physical characteristics such as color , liquefaction etc ., were observed by FT-IR

15. 4. Encapsulation efficiency The Encapsulation efficiency describes the % of the aqueous phase and hence the % of water soluble drug that becomes ultimately entrapped during preparation of liposomes and is usually expressed as % entrapment /mg of lipid drug entrapped in liposomes % encapsulation = × 100 Total drug added

16. Minicolumn centrifugation method Used for both purification and separation of liposomes on small scale.

17. Protamine aggregation method This method used for neutral and negatively charged liposomes

18. Ultracentrifugation

19. Encapsulation efficiency For estimation of EE, the un-encapsulated or free drug is separated from the microparticles by multiple washing. The microparticle free from unentrapped drug are lysed and drug content is determined using suitable analytical technique. Higher EE of drug is required for an ideal microparticle formulation and it depend on multiple factors such as method of preparation, quality, quantity of polymer, process variables viz., temperature, pH, stirring speed and time etc .

20. 5. Phase Transition Temperature

21. Light microscopy Widely used instrument

22. Phase transition temperature determined

23. 3 . Thermal analysis Thermal analysis of microparticles is done in order to investigate the physical status of the drug in the encapsulated form and also characteristics of polymer after being formulated in to the microparticles. These studies done immediately after formulation of microparticles as well as over its life time. DSC used to check solid state chemistry and physicochemical properties of the drug such as melting point

24. Thermal analysis Any alteration or disappearance in the melting point peak may indicate change of crystal structure of the API. Thermal Analysis of polymer viz., glass transition temperature can be assessed by DSC. Thermogravimetric Analysis may used to investigate phase transition, desolvation, decomposition sublimation, absorption, adsorption and desorption behavior of the polymeric microparticle.

25. 6 . Stability Studies Need to gather information on the quality of drug product over time under the influence of variety of environmental factors such as temperature, humidity and light.` Microparticle are subjected to the different accelerated and conventional stability testing procedures. Intrinsic stability of drug molecule is evaluated in the stress testing and it suggests the degradation pathways, degradation product as well as the stability indicating capability of analytical procedures employed.

26. Random selection of 3 batches. Storage condition should be 25 ± 2 °C / 60% ± 5% RH for 12 months in long term testing. 40 ± 2 °C / 75% ± 5% RH for 6 months in accelerated testing condition. Any significant change observed in the c stability testing of microparticles are further subjected to an intermediate condition e.g., 30 ± 2 °C / 60% ± 5% RH. significant change at the accelerated condition as per ICH guidelines is defined as : A 5% potency loss from initial assay value of a batch. Any specified degradant exceeding its specification limit A product exceeding its pH limit. Dissolution exceeding its specification limits Failure to meet specifications for appearance and physical properties e.g., color, phase separation, re-suspendibility, delivery per actuation, caking, hardness etc.,

27. The long term testing will be performed for sufficient time beyond 12 months to cover shelf life at appropriate test period. The testing frequency should be adequate to establish the stability characteristics of the drug product and is normally at every three months over the first year, every six months over the second year and then annually.

28. Stability Stability in vitro mainly covers the chemical stability of the constitutive lipid under the various accelerated or long term storage condition. Chemically phospholipid are susceptible to hydrolysis. Phospholipid containing unsaturated fatty acid are vulnerable to oxidative degradation and peroxidation.

29. LIPOSOME

30. 1. Lamellarity Assessed by method To determine Lamellarity Freeze Fracture Electron Microscopy 31P Nuclear Magnetic Resonance Analysis The number of bilayer present in the liposome

31. Electron Microscopic Technique Negative stain electron microscopy (EM) has been used to characterized the morphology, size and shape of the lipid particles present in phospholipid dispersion. EM also gives gross estimate of lamellarity since stain penetrates interbilayer spaces and allows lamellae to be resolved.

32. 31P Nuclear Magnetic Resonance Analysis Liposome  Lamellarity can be determined by 31P  NMR (most accurate and straightforward technique ). In this technique, the addition of metal salt (viz. Mn2+, Pr+3, Eu+2) quenches the 31P NMR signal from phospholipids on the exterior face of the liposomes. Mn2 + interacts with the negatively charged phosphate groups of phospholipids and causes a broadening and reduction of the quantifiable signal.  The degree of lamellarity is determined from the signal ratio before and after Mn2+ addition The percentage of relative loss of signal from the peak area before and after addition of Mn2. The number of bilayers is calculated by:

33. 31P NMR spectra of a) unbound b) one side Mn+2 bound c) Two side Mn+2 bound

34. Thus 50% reduction in NMR signal intensity indicates a unilamellar whereas subsequent reduction indicates a multilamellar vesicular preparation.

35. 2 . Trapped volume The internal or trapped volume is the aqueous entrapped volume per unit quantity of lipid and expressed as µl/µ mol or µl/mg of total lipid. This can vary from 0.5 µl/µ mol for some MLV and SUVs to as much as 30 µl/µ mol for certain LUVs. Material used to determine internal volume 1. radioactive marker 2. fluorescent marker

36. Best way to measure internal volume is to measure quantity of water directly and this may done by replacing the external medium ( water) with a spectroscopically inert fluid ( deuterium oxide) and then measuring the water signal using NMR The permeability of the liposomal membrane to water is such that H2O and D2O equilibrate very rapidly throughout the whole volume of the medium. The NMR scan of this medium can used to access peak height, which can be related to concentration by comparison with standards containing known amount of H2O in D2O.

37. Removing external radioactivity by Centrifugation, dialysis or gel filtration Which determined proportion of solute trapped Subsequently residual activity per lipid is determined

38. PREFORMULATION OF MICROSPHERE

39. study of surface characteristics and the internal core structure of microparticles

40. 2 . Flow properties Flow characteristics of microparticles can be determined by angle of repose, H ausners ratio, carrs index. Because of spherical shape, microsphere are expected to have good flow, but in some instances, the adhesive nature of the polymers used in the formulation may cause in poor flow as well as adhesion. Flow properties of microsphere increase by adequate drying, use of non-adhesive and non-hygroscopic material and use of glidants / lubricants / anti-adherent.

41. 3 . In vitro drug release ( Dissolution ) Determine the bioavailability of drug. Dissolution apparatus :- Basket ( USP type-1 dissolution apparatus ) or incorporated into dialysis tube and kept inside dissolution medium. Dissolution time will depend on type of formulation by maintaining sink condition. Aliquots of required volume are collected at pre specified time period and analyzed by suitable analytical method.

42. In vitro drug release ( Dissolution ) Alternatively microsphere loaded with API are suspended in dissolution medium in screw capped tubes and placed in orbital shaker maintained at 37±1°C Tubes are removed at pre-decided time interval and subjected to centrifugation. Then supernatant is removed and remaining microsphere are resuspended in fresh dissolution medium and placed back in shaker. Supernatant containing drug is analyzed by suitable analytical method.

43. 4 . Ex vivo drug permeation Microparticle are intended for transdermal or topical application The skin of animal ( rat, goat or pork ) is fixed at junction of donor and the receptor compartment of Franz diffusion cell. The study is performed with F ranz diffusion cell, Keshri chain diffusion apparatus or flow through diffusion cells. Microparticle put on the skin surface. Put in require dissolution medium and temperature is maintained at 37±1°C The dissolution medium is stirred with the help of magnetic stirrer. The API permeated in the dissolution medium through the biological membrane is collected through the sampling probe and subjected to evaluation by suitable analytical technique.

44. REFERNCE Jain S., Jain N. ”liposome as drug carriers” controlled and novel drug delivery, CBS publishers and distributors Int . J. Drug Dev. & Res . | October - December 2013 | Vol. 5 | Issue 4 | ISSN 0975-9344 | https://books.google.co.in/books?isbn=0849340128 Yechezkel Barenholz, ‎Danilo D. Lasic - 1996 – http://scinel.blogspot.in/2016/01/31p-liposomal-lamellarity.html