liposomal gene delivery updated.pptx

Liposomal gene delivery

1. Topic- Liposomal gene delivery Submitted to – Dr. Rekha Rao mam Submitted by – Sheetal Sardhna

2. LIPOSOMES Liposomes are artificial spherical vesicles which contain a small volume of aqueous solution enclosed in a lipid bilayer. Liposomes consist mainly of phospholipids and cholesterol, major components of biological membranes. Liposomes are most often composed of phospholipids , especially phosphotidylcholine and cholesterol which are major components of biological membranes. 2

3. LIPOSOMES AND CELL MEMBRANE INTERACTION The cell membrane is composed mainly of lipids and proteins. Liposome and cell membrane interactions have been studied as a model of biological membrane interactions. There are four basic types of interaction between liposomes and cell membrane; Inter-membrane transfer mediated lipid transfer between liposome and cell membrane. 3

4. Fig: Liposomes and cell membrane interaction 4

5. Continue.. 2. Liposomal adsorption can be enhanced by modification of lipid bilayer, with antibodies against cell surface antigens, various receptors, or giving electric charge to liposome. 3. Some of the liposomes fuse with endosomal membrane to release the entrapped substances directly into the cytoplasm, such as low pH sensitive liposome. 4.Through the fusion process, entrapped materials are directly released into the cytoplasm and some of the plasmids are transferred into the nucleus. 5

6. INTERMEMBRANE TRANSFER With transfer of liposomal lipids to cellular and subcellular membranes or vice versa 6

7. ADSORPTION TO THE CELL MEMBRANE Adsorption to the cell surface is done either by non specific weak hydrophobic or electrostatic forces , or by interactions of specific receptors on cell membrane surface to ligands on the vesicle membrane. For water soluble components, vesicles contents are diffused through the lipids of the cell. For lipids soluble components, vesicles contents are exchanged with the cellular membrane along with the lipid of the vesicle. 7

8. FUSION WITH THE CELL MEMBRANE Fusion with the cell membrane is by insertion of the lipid bilayer of the liposomes into the cell membrane to become part of the cell wall, with simultaneous release of liposomal content into the cytoplasm. 8

9. LIPOSOMAL GENE DELIVERY In gene therapy, genetic materials, such as plasmid DNA, antisense oligonucleotides ( asODNs ), and small interfering RNA (siRNA), can be used to treat or prevent disease. This includes replacing a mutated gene, inactivating a mutated gene, or introducing a new gene. Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections). There are several nonviral gene carriers, such as lipids, polymers, and peptides, that can be used for this purpose. Liposomal delivery of nucleic acids, such as plasmid DNA, asODNs , and siRNAs, represents a very promising nanocarrier system that is relatively safe and effective in delivering genes to targeted locations in the body. Lipoid-based delivery systems have also been shown to be stable in serum and plasma, have improved biodistribution , prolonged circulation half-life, and enhanced target tissue selectivity. The most common lipids used in liposomes are cationic lipids, which facilitate binding between their positively charged head group(s) to negatively charged nucleic acids. 9

10. CHARACTERSTICS OF LIPOSOMES Liposomes are spherical vesicles comprised of phospholipids, forming one or more bilayers around an aqueous solution. Phospholipids are composed of two hydrophobic fatty acid tails attached to a glycerol unit, which are linked to a hydrophilic phosphate group with a polar head molecule. When dissolved in an aqueous solution, the lipids spontaneously arrange themselves so the hydrophobic tails face each other and the hydrophilic head groups face towards the interior and exterior aqueous solution. The liposomes can therefore easily encapsulate hydrophilic drugs into the aqueous cores or, if the drug is hydrophobic, it can be dissolved into the membrane and in this way liposomes can carry both hydrophobic and hydrophilic drugs to the site of action. 10

11. CATIONIC LIPIDS Lipids with a positively charged head group, so-called cationic lipids, are typically used to facilitate the encapsulation of nucleic acids because the positively charged head group spontaneously causes electrostatic binding to the negatively charged nucleic acids. The positive charge also enhances entry into cells by electrostatic attractions to the slightly negatively charged cell membrane. Commercially available transfections reagents all contain cationic lipids that, when mixed with, for example; plasmid DNA, form a positively charged complex; a lipoplex( mid-cell structure) For example: DOTMA-N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trchlorilammonium chloride DOTAP-[1,2-bis( oleoyloxy )-3-( trimethylammonio )propane] 11

12. Continue.. ANIONIC LIPIDS- Using anionic lipids to encapsulate nucleic acid is quite inefficient. The negatively charged head group causes electrostatic repulsion of the negatively charged nucleic acids. To solve these problems, physiological cations, such as calcium (Ca2+), can be added, causing complexation between calcium and the nucleic acids. For example: Phosphotidylserine , Phosphatidylglycerol NEUTRAL LIPIDS- Neutral lipids and polymers are hydrophobic molecules that lack charged groups and can therefore not condense nucleic acids by means of electrostatic interactions . For example: DOPE- dioleoylphosphatidylethanolamine DOPC- dioleoylphosphatidylcholine 12

13. STEPS IN LIPOSOMAL GENE DELIVERY Liposomal gene delivery typically involves four steps: Formulation Administration Uptake Expression 13

14. Fig: liposomal gene delivery 14

15. 15 HMG – HYDROXYMETHYLGLUTARYL- COENZYME A REDUCTASE INHIBITOR INACTIVATED HVJ- HEAMGLUTTINING VIRUS OF JAPAN – A MEMBER OF MOUSE PARAMYXOVIRUS FAMILY HAS BEEN COMBINED WITH LIPOSOMES TO PRODUCE A NOVEL GENE TRANSFER SYSTEM NAMELY HVJ LIPOSOMES

16. STEPS IN LIPOSOMAL GENE DELIVERY Formulation: liposomes are prepared by combining cationic and anionic lipids in a specific ratio. The cationic lipids are positively charged and interact with the negatively charged nucleic acids such as DNA & RNA. The anionic lipids provide stability and help to prevent the liposomes from fusing with each other. Other components such as Cholesterol and PEGlyted lipids can be added to improve the stability and efficacy of the liposomes. Administration: liposomal gene delivery can be administered in several ways including Injection , Inhalation or topical application . The method of administration will depend on the target tissue and the disease being treated. 16

17. Continue.. Uptake: Once the liposomes are administered they must be taken up by the target cells. This can occur through several mechanism including endocytosis, fusion, or direct uptake. The cationic lipids in the liposomes interact with the negatively charged cell membrane, allowing the liposomes to fuse with the cell membrane or enter the cell via endocytosis. Expression: once the therapeutic genes is inside the cell, it must be expressed to have a therapeutic effect. The gene can integrate into the host genome, or it can remain episomal and can be expressed transiently . The gene may also need to be activated by a specific promoter or inducer to ensure expression in the target tissue. 17

18. MODIFICATIONS IN LIPOSOMAL GENE DELIVERY POLYETHYLENE GLYCOL: Recent improvements in lipofection have facilitated protection from degradation in vivo, due to surface modifications with polyethylene glycol (PEG). It was shown by “Kim et al” that PEGylated lipoplex yield, increases transfection efficiencies in the presence of serum as compared to liposomal transfection. Additionally the PEGylated lipoplexes display improved stabilities and longer circulation times in blood. PEG forms a steric barrier around lipoplex and results into reduced aggregation and increased bioavailability. PEG presents many attractive qualities as a liposomal coating, such as: availability in a variety of molecular weights, Lack of toxicity., Ready excretion by the kidneys. These particles are sometimes referred to as “stealth liposomes.” 18

19. CONT… ADDITIONS AND ALTERNATIVES TO POLYETYLENE GLYCOL- Alternatives liposomal formulation formulations utilizing polymers other than PEG are being produced with a goal of creating sterically protected lipoplexes. Additional aims of such systems include biocompatibility , flexible structure, and solubility in physiological systems. A report by Metselaar et al. on the use of L-amino acid based polymers for lipoplex modification found an extended circulation time and reduced clereance by macrophages at levels similar to those seen with lipoplexes modified with PEG. Results suggested that approximately 10% of the injected dose of the L-amino acid modified complexes was still present in the blood of treated rats after 48 hours. These oligopeptides are attractive alternatives to PEG due to advantages such as increased biodegradeability and favorable pharmacokinetics when lower concentations are used per dose. 19

20. ADVANTAGES Simplicity.
Non-infectious..
Long term stability
Low degree of toxicity.
Protection of nucleic acid from degradation.
Easy to manipulate and use. Economical Low cost Lack of immunogenic response Provides nuclease protection and targetability Efficient delivery of nucleic acids to cells in a culture dish It has no constraints on the size of the gene that has to be delivered 20

21. DISADVANTAGES Targeting is not specific Low transfection efficiency
Only transient expression
Difficult in-vivo application Not applicable to all types of cell 21

22. FUTURE PROSPECTS FOR LIPOSOME-MEDIATED GENE THERAPY For in vivo gene therapy using liposome-mediated gene transfer, much effort is being exerted to develop methods for specific targeting of particular organs, increasing efficient uptake of entrapped genes into cytoplasm, targeting of genes into nucleus, and achieving long-term gene expression. With the HVJ-liposome method , we can target organs by changing the route of administration , but more precise organ targeting may be accomplished through by liposome. We can prepare immunosomes and virosomes by putting immunogen or viral protein into the lipid bilayer. 22

23. To enhance gene transfer into the cytoplasm , HVJ fusion activity is utilized. For long term genes expression , it is important to stabilize , regulate and replicate transgenes. 23

24. FDA APPROVED DRUGS FOR LIPOSOMAL GENE DELIVERY Trade name Active ingredients Application ABELECET Amphotericin –B Fungal infection DAUNOXOME Daunorubicin Kaposi’s sarcoma DEPOCYT Cytarabine Neoplastic meningitis Lymphomatous meningitis DOXIL Doxorubicin Kaposi’s sarcoma Ovarian cancer Breast cancer LIPOPLATIN/NANOPLATIN Cisplatin Pancreatic cancer 24

25. ANTIGENICITY AND TOXICITY Although liposomes consist mainly of biodegradable lipids, liposome toxicity and antigenicity must always be a concern in the development of clinical products. Cholesterol, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol have no effect on the synthesis of the DNA in the cells, whereas, stearylamine (SA), phosphatidylserine and dicetyl phosphate (DCP) reduce DNA synthesis. DCP and SA have also been reported as toxic compounds when injected intra cerebrally into mouse brain. 25

26. BARRIERS IN LIPOSOMAL GENE DELIVERY Systemic Barriers - After systemic administration of liposomes containing genetic material, liposomes should stay intact in the blood, have little or no interaction with serum proteins, erythrocytes and other cellular components and be able to reach the target tissue. Cellular barriers- When liposomes reach a target cell, it has to overcome certain barriers for successful transfection. These barriers include (a) binding of the liposome to the cell surface , (b) entry of the liposome into the cells by endocytosis or direct traversing of the plasma membrane (e.g. via membrane fusion), (c) escape of the liposome from the endosome, (d) dissociation of the liposome to release nucleic acid payload, (e) transport through the cytosol and (f) entry into the nucleus 26

27. Barriers of liposomal gene delivery 27

28. REFERENCES Review article of “ Gene delivery by liposomes” Written by Mirjam M. Nordling -David and Gershon Golomb Published by Wiley online library in Israel Journal of chemistry. Review article of “Liposome mediated gene transfer ” written by Itaru Yanagihara , Yasufami Kaneda, Koi Inui and Shint Aro Okada Published by chapman and hall in Molecular and Cell Biology of Human Gene Therapeutics. Review article of “Liposome for used in gene delivery ” written by Daniel A. Balazs and WT. Godbey Published by Hindawi publishing corporation in Journal of Drug delivery 28

29. THANK YOU 29