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
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2. CONTENTS
■ LIPOSOMES
■ GENE THERAPY
■ LIPOSOMAL GENE DELIVERY SYSTEMS
- INTRODUCTION
- PRINCIPLE
- CHARACTERISTCS
- CLASSIFICATION OFLIPIDS
- ADVANTAGES
- DISADVANTAGES
- USES
■ CONCLUSION
■ REFERENCES
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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
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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.
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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: . :
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GENE
THERAPT
SOMATIC
GENE
THERAPY
EX VIVO
IN VIVO
GERM LINE
GENE
THERAPY

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
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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.
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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
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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.
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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 .
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LIPIDS
CATIONIC
LIPIDS
MONOVALENT
cl
MONOVALENT
cl
NEUTRAL
LIPID
ANIONIC LIPID

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