Liposomes: A novel drug delivery

The size of the market for delivery of liposome-based medicines depends on the growing prevalence of chronic diseases and the growing demand for non-invasive drug distribution solutions.In 2019; the liposomal doxorubicin sector accounted for around 36.22 percent of the market.In 2021, the cancer therapy segment represented the greatest share of the market in terms of application.
The market is estimated to grow at a CAGR of 8.8% from 2020 to 2027.

1. LIPOSOMES: A DRUG DELIVERY SYSTEM Presented By- Under The Guidance of:- Ms. Vinayshri S. Salunkhe. Dr. H.S.Mahajan. Roll No:- MPH-14 Dept. of Pharmaceutics 04-08-2021 1

2. Contents- Background Introduction Classification of liposomes Methods of preparation Interaction of liposomes with cells Application of liposomes Characterization of Liposomes Current liposomal drug preparation Conclusion References 04-08-2021 2

3. Global Liposome Drug Delivery Market: Introduction T he global  liposome drug delivery market   was valued at  US$ 3.6 Bn in 2018  And it reach valuation of ~US$ 8 Bn By 2027 04-08-2021 3 https://www.transparencymarketresearch.com/liposome-drug-delivery-market.html

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5. Introduction - “ Liposomes(Lipos-fat; Soma- body) are concentric, self-closed [4] , microscopic vesicle in which an aqueous volume is entirely enclosed by curved membranous lipid bilayer [1] and the drug molecules can either be encapsulated in aqueous space or intercalated into the lipidic bilayers [2] .” -Carry both hydrophilic and lipophilic molecules. Fig.1 : General structure of liposome 04-08-2021 5

6. Components of liposome structure- Phospholipids and cholesterol- main components. Phospholipids – - Amphipathic and capable of forming bilayer hence are integral part of liposomes [4] . -2 acyl chains linked to a head group by means of glycerol -backbone [4] . -Most common phospholipid is Phosphatidylcholine (PC ). Known as “lecithin” [2] . -At various temperature it exist in different phases. Fig.2: Different regions of liposome 04-08-2021 6

7. Cholesterol- - Provide rigidity to fluid phase vesicle. It act as a “fluidity buffer” [3] . -Itself does not form bilayers, but it can be incorporated in 1:1 or 2:1 molar ratio, can bring major changes in membrane [3] . - Increases the Tc of the membrane & decreases the permeability of the bilayer [3] . - Restrict the transformation of trans to gauche confirmation [3] . - Transition temperature of phospholipids(Tc) (Temperature at which all lipids changes their fluidity ) It determines fluidity and permeability of bilayer & influence the curvature of liposomes [5] . Temp <T C lipids are gel [5] . Temp >T C Lipids are in liquid-crystalline phase [5] . Longer chain at higher T C [5] . -Tc depends on length of fatty acid chain , their degree of saturation charge and head group species [3] . 04-08-2021 7

8. Classification of liposomes [3] - Types Based on structural parameter Multilamellar large vesicles (>0.5 µm) Oligolamellar vesicles (0.1-1 µm) Unilamellar vesicles (all size range) Small unilamellar vesicles (20-100nm) Large unilamellar vesicles (>100nm) Based on composition and application Conventional liposomes(CL) pH sensitive liposomes Cationic liposomes Long circulatory (stealth) liposomes(LCL) Immuno-liposomes 04-08-2021 8

9. Conventional Long circulating Immuno Cationic Fig.3 Classification of liposomes 04-08-2021 9

10. Methods of Preparation [3] - Methods of liposome preparations Passive loading techniques Active loading techniques Mechanical dispersion methods Solvent dispersion methods Detergent removal methods Liquid film hydration by -Hand shaking, -non-hand shaking Sonication French pressure cell Membrane extrusion Micro-emulsification Dried reconstituted vesicles Freeze-thawed liposomes Ethanol injection Ether injection Double emulsion vesicles Reverse phase evaporation vesicles Detergent removal from mixed micelles by - Dialysis - Column chromatography - Detergent adsorption using Bio-beads Size Reduction 04-08-2021 10

11. General method of liposome preparation [3] - 04-08-2021 11 Drying down lipid from organic solvent Dispersion of lipid in aqueous medium Purification of resultant liposomes Analysis of final product

12. 1.Mechanical dispersion method - Lipid dissolve in organic solvent/co-solvent Remove organic solvent under vacuum Film deposition Solid liquid mixture is hydrated by using aqueous buffer Lipid spontaneously swell & Hydrated Liposome formation A) Lipid hydration method [2,3] - Hand shaking method Non Hand shaking method Dispersion of film by manual agitation Provide agitation by rotary flash evaporator by exposing film to a steam of nitrogen Multilamellar Vesicles Large Unilamellar Vesicles Encapsulation efficacy as high as 30% Encapsulation efficacy high Passive loading technique [3] 04-08-2021 12

13. a)Sonication- Size reduction of multilamellar vesicles [3] - To convert large size into smaller homogeneous vesicles. Which includes following techniques but second set of method to increase entrapment volume of hydrated lipids/to reduce lamellarity freeze-drying, freeze thawing or introduction of vesiculation by ions or pH change [3] . MLV in test tube Sonicate for 5-10 min above phase transition temperature Filter &centrifuge at 100000 rpm for 30min at 20℃ Decant top layer Sonicated unilamellar vesicles 04-08-2021 13

14. b) French Pressure Cell [3] – The extrusion of MLV at 20,000 psi at 4℃ through a small orifice. Yields Uni- or oligo- lamellar vesicles (30-80 nm) Advantages over sonication method. -More stable, less structural defect -leakage of content is lower than sonication c) Membrane extrusion [3] – Size of liposome reduced by passing them through membrane filter. Much lower pressure (<100psi) 04-08-2021 14

15. B) Micro-emulsification [3] – -Micro fluidizer pumps the fluid at high pressure (10,000psi,600-700bar) through a 5µm orifice. -Negative lipids tends to decrease their size, increasing cholesterol concentration gives larger liposomes. 04-08-2021 15

16. D) Freeze-thawed liposomes [3] - C) Dried-reconstituted Vesicles [3] - 04-08-2021 16

17. 2) Solvent dispersion methods [2,3] - Ethanol injection- - Alternative for preparation of SUV without sonication. - low risk of degradation of sensitive material b) Ether injection – - It has little risk of causing oxidative degradation -careful control needed for introduction of lipid solution 04-08-2021 17

18. c)Double emulsion [3] – Organic solution + Lipid + Aqueous phase Emulsion(w/o) Hot aqueous solution of buffer Multi lamellar vesicle w/o/w(double emulsion ) LUVs d ) Reverse phase evaporation(MLV,LUV) [3] - Emulsion Evaporation under reduced pressure, rotary evaporator Semi solid gel Shake to get LUVs 04-08-2021 18

19. Dialysis Column chromatography Detergent Adsorption using Bio-beads- 3) Detergent depletion/solubilization method of passive loading [3,4] Detergent removed from mixed micelle by dialysis, which is facilitated by using high CMC. Ex: Sodium cholate, Sodium deoxycholate, octyl glucoside. Removal of deoxycholate by passing the dispersion over Sepadex G-25 column pre-saturated with constitutive lipids and pre-equilibrated with hydrating buffer. Removal of detergent by using proper adsorbent. Ex: Bio-beads SM-2 used to adsorb Triton X-100. 04-08-2021 19

20. Active loading technique [3] After drying in process Film/cake of lipid is form Swelling in fluid Formation of liposomes Loading of drug on pH- Gradient technique 2 process which causes pH imbalance and active loading : 1. Vesicles are prepared in low pH solution, thus generating low pH within the interiors. 2. Addition of base to extra liposomal medium. 04-08-2021 20

21. Mode of liposome/Cell interaction- 04-08-2021 21

22. Characterization of Liposomes [2,3,4] : Parameters Techniques used Shape, Lamellarity Freeze-fracture and Freeze-etch electron microscopy [3] NMR [3] Atomic force microscopy [4] Size and size distribution Laser light scattering [2] (Dynamic light scattering, turbidity measurement) TEM(Negative stain, Cryo-transmission) [3] SEM [3] Gel permeation [2,3] Surface charge Capillary zone electrophoresis [4] Zeta potential [3] Encapsulation efficiency & Entrapped volume Minicolumn centrifugation [3,4] Using Radioactive markers [3,4] Gel filtration, Dialysis [4] Protamine aggregation [3,4] Phase transition temperature DSC [4] , NMR [4] Release Dialysis 04-08-2021 22

23. Recent liposomal drug preparations Type of Agents Examples Anticancer Drugs Daunorubicin(Dauno xome ® ), Doxorubicin(Doxil ® , Rubilong ® ) , Paclitaxel(Taxol ®) Anti bacterial Ampicillin(Amfight ®) , piperacillin, rifampicin Vaccines Corona virus vaccine (Pfizer, Moderna) Hepatitis A antigen(Epaxal-Berma vaccine ®) , Influenza (Influsome-Vac ® ) Rabies virus, Malaria merozoite, Malaria sporozoite, Antiviral AZT (Retrovir ®) Fungicides Amphotericin-B(AmBisome ®) Enzymes Hexosaminidase A ,Glucocerebrosidase, Peroxidase Pain Management Morphine(DepoDur ®) Gene therapy mRNA vaccine (Pfizer, Moderna) 04-08-2021 23

24. Liposomal preparation in current scenario - Liposomal Amphotericin-B –: Black fungus infections.(~10lakh vial allocated across the country). mRNA vaccines of Moderna and Pfizer/BioNTech-: Corona virus infection 04-08-2021 24

25. Conclusion- 04-08-2021 25 Liposomes having multiple advantages which includes increased stability of the encapsulated drug, reduced contact of sensitive tissues with therapeutic molecules, decreased drug toxicity, improved pharmacokinetic and pharmacodynamics properties, the ability to regulate the rate of drug release, and the potential of their structure to accept the desired chemical modification. Hence, The demand of liposome within the global liposomes market is raised on account of advancements in medical and pharmaceutical research. So, by reviewing liposomes pros and cons, scientists will be able to improve them in future research works and meet the global demand .

26. Bangham A.D, Standish M.M. Watkins, J.C. “The First Description of Liposomes” J. Mol. Biol., 13:238-252 (1965). N.K. Jain, “Controlled and Novel Drug Delivery”, 1 st ed.’ CBS Publishers and Distributors, New Delhi ( 1997) , pp. 305-344. S.P. Vyas and R. K. Khar, “Targeted and Controlled Drug Delivery: A Novel Carrier System”, 1 st ed; CBS Publishers and Distributors, New Delhi ( 2002) , pp. 173-243. Dr. R. S. R. Murthy, “Vesicular and Particulate Drug Delivery System”, 1 st ed; Career Publications, Maharashtra ( 2010) , pp. 5-73. Higuera-Ciapara, Beltran-Gracia, E.,Lopez-Camacho,A., “Nanomedicine review: clinical developments in liposomal applications”. Cancer Nano 10, 11 (2019 ). Rommasi,F., Esfandiari, N. “Liposomal Nanomedicine: Applications for Drug Delivery in Cancer Therapy”. Nanoscale Res Lett 16, 95 (2021) . References- 04-08-2021 26

27. 7. Yang Y, Yang X, Li H, Li C, Ding H, Zhang M, Guo Y, Sun M. Near-infrared light triggered liposomes combining photodynamic and chemotherapy for synergistic breast tumour therapy. Colloids Surf B Biointerfaces. ( 2019) Jan 1;173:564-570. 8. Abeyratne E, Tharmarajah K, Freitas JR, Mostafavi H, Mahalingam S, Zaid A, Zaman M, Taylor A. Liposomal Delivery of the RNA Genome of a Live-Attenuated Chikungunya Virus Vaccine Candidate Provides Local, but Not Systemic Protection After One Dose. Front Immunol. ( 2020) Mar 5;11:304. 04-08-2021 27

28. Thank you 04-08-2021 28