Liposomsl gene delivery SYSTEM

LIPOSOMES gene therepy liposomal gene delivery system classsification of lipids

1. LIPOSOMAL GENE DELIVERY SYSTEMS Presented By Sujitha Mary M Pharm St Joseph College Of Pharmacy 1

2. CONTENTS LIPOSOMES GENE THERAPY LIPOSOMAL GENE DELIVERY SYSTEMS INTRODUCTION PRINCIPLE CHARACTERISTCS CLASSIFICATION OFLIPIDS ADVANTAGES DISADVANTAGES USES CONCLUSION REFERENCES 2

3. LIPOSOMES These are the concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membrane lipid bilayer mainly composed of natural and synthetic phospholipids. Its Size range varies from 20nm-3 microns. Liposomes are spheres of lipids which can be used to transport the molecules into the cells 3

4. Cholesterol is often essential to achieve sufficient stability of these liposomes and act as fluidity buffer DRUG PROPERTIES AND LIPOSOMAL ASSOCIATION : . Hydrophilic Retained in aqueous interior,but may be difficult to get high drug loading. Slowly released over several hours- several days. Hydrophobic Inserted into hydrophobic interior of liposomal bilayer, but can disrupt liposome at higher concentration. Excellent retention. Intermediate Rapidly partition between lipid bilayer and aqueous phase Rapid release from liposomes but pH manipulation or change in formulation of molecular complex can result in good retention. 4

5. GENE THERAPY Gene therapy is an experimental technique that uses genes to treat or prevent diseases by introducing DNA into cells:  It is an alternative therapy of using drugs or surgery. TYPES Gene therapy is the one which is currently being tested for the treatment of diseases that has no other cures But genetic materials are high molecular weight polar compounds that don’t permeate the biological barrier easily and it requires special carriers and targeting methods. So, these gene delivery systems were categorised as follows: . : 5

6. Key to success for any gene therapy strategy is to design a vector that is able to serve as safe and efficient gene delivery vehicle which encouraged the development of non-viral DNA mediated gene transfer technique. Viral vectors .  These have stability and are very efficient in transducing cells but their cost is high and toxicity is a big issue. Non-viral vectorsThese are safe and non-invasive delivery methods and easy to handle 6

7. LIPOSOMAL GENE DELIVERY SYSTEMS: INTRODUCTION The encapsulation of plasmid DNA into liposomes and the introduction of poliovirus RNA and SV40 DNA into cells via liposomes were reported between 1979 and 1980. These are vesicles that can easily merge with the cell membrane. since, they both are made up of phospholipid bilayers , and these are surrounded by the molecule to be transported and promote its transport after fusing with the cell membrane The technique which is used to inject the genetic material into a cell by means of liposomes is known as “ Lipofection or liposome transfection or lipoplex ”. Lipoplex are non-viral synthetic nucleic acid carriers utilizing cationic phosphate lipid mixtures to condense and protect the nucleic acid. PRINCIPLE: Lipofection generally uses a positively charged(cationic)lipid to form a structure with the negatively charged(anionic)genetic material. Fusion of the liposome/nucleic acid transfection complex with the negatively charged cell membrane takes place. The transfection complex is then enter into the cell through endocytosis . 7

8. Once enter inside the cell ,the complex must escape the endosomal pathway, diffuse through the cytoplasm and enter the nucleus for gene expression. E.g.,Animal cells, plant cells,Bacteria,yeast protoplast are susceptible to lipofection method CHARACTERISTICS: Liposomes are generally formed by the self-assembly of dissolved lipid molecules, each of which contains a hydrophilic head group and hydrophobic tails and these can exhibit a range of sizes and morphologies upon the assembly of pure lipids or lipid mixtures suspended in an aqueous medium.. During the compaction of polynucleotides into liposomes assemblies number of structures have been known to appear. Each structure is formed in the most energetically favourable conformation based upon the specific lipids used in the system 8

9.   A dependent term known as “structure packing parameter” which can be used to suggest what shape the amphiphile will take depending on the ratio of size variables The packing parameter is defined as: P=V/ alc where,v =volume of hydrocarbon portion. a=effective area of head group lc =length of lipid tail. This correlation predicts a range of structures According to the following conditions. 9 Packing parameter structures <1/3 spherical micelle 1/3≤p<1/2 cylindrical micelle ½≤p<1 flexible bilayers ,vesicles p=1 planarbilayers P>1 inverted micelle

10. But here, the encapsulation of DNA into conventional liposomes could be a terminal problem due to the plasmid size which results into poor transfection . Alternative technology is the formation of cationic liposomes and PE complex which was developed in late 1980’s Based on the charge on head group in lipoplex , these lipids were categorised into 3 types . 10

11. 1)CATIONIC LIPIDS Cationic lipids are those having a positive charge which associate with the negatively charged DNA molecules by electrostatic interactions, forming a stable complex and are used for the transfer of nucleic acids. Cationic head group appear to be better suited for DNA delivery due to the neutral charge attraction between negatively charged phosphate group and positively charged head group. 11

12. Its idea is to neutralize charge of plasmid with positively charged lipids to capture plasmid more efficiently and to deliver DNA into cells.   The basic structure of cationic lipids mimics the chemical and physical attributes of biological lipids   Here, lipoplex is formed by combining the cationic lipids, neutral lipids and genetic material  Cationic lipids should be chemically stable, biodegradable and protect against degradation of DNA by cell.. Cationic lipids are divided into 2 TYPES. A) MONOVALENT CATIONIC LIPIDS: i ) DOTMA: It was one of the first synthesized cationic lipid and was described by “ felgner et al” Its structure consist of 2 unsaturated oleoyl chains(c18;Δ9)bond to an ether bond to the 3-carbon skeleton of a glycerol with a quaternary amine as cationic head group. DOTMA proved to facilitate up to 100 fold more efficient gene expression then the use of DEAE- dextran co- ppt or ca2po4 . Liposomal sensitivity is 25%-30%.. 12

13. PROCEDURE: Mix cationic lipids+ neutral lipids+ DNA to cell results in the formation of aggregation composed of DNA and cationic lipids. DOTMA either alone or in combination with other neutral lipids spontaneously form MLV which may be further sonicated to form SUV. DOTMA ( lipofective TM) can reduce cytotoxicity. 13

14. ii)DOTAP: Its structure consist of a quaternary amine head group coupled to glycerol backbone with 2-oleoyl chains Difference between DOTMA and DOTAP are that ester bonds link the chains to the backbone rather than ether bonds Liposomal sensitivity is 25%-30%.. But DOTAP is completely protonated at pH(7.4). so, that more energy is required to separate the DNA from the lipoplex for successful transfection . Thus, for DOTAP to be more effective in gene delivery it should be combined with a helper lipid as seen in most cationic lipid formulation. In this, high temperature and long incubation time have been used to create lipoplex that exhibit resistance to serum interaction. . 14

15. iii)DC-CHOL It contains a cholesterol moiety attached by an ester bond to a hydrolysable dimethyl ethylene diamine . It was first synthesized by “GAO and HUANG” in 1991 . Cholesterol is chosen for its biocompatibility and stability and it imparts to lipid membrane B)MULTIVALENT CATIONIC LIPIDS : i)DOSPA:. It is similar to DOTMA except for a spermine group which is bound via a peptide bond to a hydrophobic chain : This cationic lipid, used with a neutral helper lipid DOPE at a 3:1 ratio which is commercially available as the transfection regent “LIPOFECTAMINE 15

16. ii)DOGS: It structure is similar to DOSPA as both molecules have a multivalent spermine head group and two 18 carbon alkyl chains.  However,the chains in DOGS are saturated and are linked to the head group through a peptide bond and lack a quaternary amine DOGS is commercially available under the name “TRANSFECTAM ”This lipid has been used to transfect many cell lines, with transgene expression levels more than 10-fold greater than those seen in calcium phosphate transfections In addition, “Behr et al” showed that not only DOGS was very effective in delivering the CAT reporter plasmid, but it was also associated with no noticeable cytotoxicity 16

17. MODIFICATIONS: POLY ETHYLENE 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. ease of application :These particles are sometimes referred to as “stealth liposomes .” 17

18. Additions and Alternatives to Polyethylene Glycol: Alternative liposomal formulations utilizing polymers other than PEG are being produced with the goal of creating steric ally 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 clearance by macrophages at levels similar to those seen with lipoplexes modified with PEG. These oligopeptides are attractive alternatives to PEG due to advantages such as increased biodegradability and favourable pharmacokinetics when lower concentrations are used per dose 18

19. 2)NEUTRAL LIPIDS Most liposomal formulations used for gene delivery consist of a combination of charged lipids and neutral helper lipids. The neutral helper lipids used are often dioleoylphosphatidyl ethanolamine (DOPE), which is the most widely used neutral helper lipid, or dioleoylphosphatidyl choline (DOPC). Neutral lipids stabilize the complex in serum and reduce toxicity Results have shown that the use of DOPE versus DOPC as the helper lipid yields higher transfection efficiencies in many cell types. 19

20. 3)ANIONIC LIPID: In general, gene delivery by anionic lipids is not very efficient. The negatively charged head group prevents efficient DNA compaction due to repulsive electrostatic forces that occur between the phosphate backbone of DNA and the anionic head groups of the lipids However , due to the fact that cationic liposomes can be inactivated in the presence of serum, are unstable upon storage, and exhibit some cytotoxicity both in vitro and in vivo .. Commonly used lipids in this category are phospholipids that can be found naturally in cellular membranes such as phosphatidic acid, phosphatidylglycerol , and phosphatidylserine Little toxicity, compaction with body fluids ,complicated and time consuming process. DOPE DOPC. 20

21. DNA entrapment in anionic liposomes is still inefficient, and cytotoxicity data remain inadequate. Sometimes , the DNA molecules get entrapped within the aqueous interior of these liposomes Then, divalent cations are used (e.g. Ca2+, Mg2+, Mn2+, and Ba2+) which can neutralize the mutual electrostatic repulsion .. They are termed as pH sensitive due to destabilization at low pH . Due to reduced toxicity and interference from serum proteins, pH-sensitive liposomes are considered as potential gene delivery vehicles than the cationic liposomes . Phosphatidic acid Phosphatidyl glycerolPhosphatidyl serine 21

22. ADVANTAGES: It has no constraints on the size of the gene that has to be delivered . Efficient delivery of nucleic acids to cells in a culture dish. Provides nuclease protection and targetability . Low cost, economical. Lack of immunogenic response. Easier to prepare than viral vectors. Easy to manipulate and use. Protection of nucleic acid from degradation. Low degree of toxicity. Long term stability Non-infectious. Simplicity . 22

23. DISADVANTAGES: Not applicable to all types of cell . Difficult in-vivo application. only transient expression. Low transfection efficiency. Targeting is not specific. 23

24. USES: Liposomes composed of DOPE/OLEIC ACID/CHOL are capable of transfecting mouse LLK-cells(cells lacking thymidine kinase with exogenous Tk gene.   Cationic lipids prepared from DOPE and DC-CHOL were reported to be significantly enhance the growth inhibitory effect of antisense oligodeoxynucleotides (ASODN)against the human telomerase transcriptase on human cervical adeno carcinoma cell both invitro and in vivo. cationic liposomes are used to deliver CDNA of cystic fibrosis trans membrane conductance regulator(CFTR) to epithelial tissue of respiratory system which is currently in clinical trails 24

25. CONCLUSION: The shape of liposomes that are formed are determined by the types of lipids used, which, in turn, provides various options regarding delivery. The cationic head group appears to be better suited for DNA delivery while anionic head groups are perhaps better suited for drug delivery One must keep in mind all of the variables that come into play when using different gene delivery vectors. There is no concrete comparison that can easily be made to suggest that one liposomal vector is better than another for all cell types, environment, and applications While some of the lipids presented above were originally found to yield little to-no toxicity for a given cell type, this information doesn't hold true when applied to different cell types Improvements and adjustments of the formulations are constantly being explored through the addition of different lipids, targeting molecules or shielding moieties designed to prevent clearance in vivo .. 25

26. REFERENCES: J. H. Felgner , R. Kumar, C. N. Sridhar et al, “Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations,” Journal of Biological Chemistry, vol.269,no.4,pp.2550-2561,1994 . C. Tros de Ilarduya , Y. Sun, and N. Duzunes , “Gene delivery by lipoplexes and polyplexes ,” European Journal of Pharmaceutical Sciences, vol. 40, no.3, pp. 159-170, 2010. T.Montier , T. Benvegnu , P.-A. Jaffres , J.-J. Yaouanc , and P.Lehn , “Progress in cationic lipid-mediated gene transfection : a series of bio inspired lipids as an example,” Current Gene Therapy, vol.8,no.5,pp.296-312,2008. A . Jesorka and O. Orwar , “ Liposomes : technologies and analytical applications,” Annual Review of Analytical Chemistry, vol. 1, pp. 801-832, 2008. Daniel A. Balazs and WT.Godbey , “ Liposomes for use in Gene Delivery,” Review Article, Hindawi Publishing Corporation, Journal of Drug Delivery, vol. 1, pp.1-12, 2011. 26