Liposomes

1. LIPID-BASED NANOPARTICLES:
LIPOSOMES
SOLID LIPID NANOPARTICLES
LIPID MICELLES
LIPOPLEXES

2. • LIPOSOMES are the smallest round structure
technically produced by natural non-toxic
phospholipids and cholesterol.
– They can be used as drug carriers and they can be
“loaded” with a huge variety of molecules, as small
drug molecules, proteins, nucleotides even plasmids or
particles.
– They have a very versatile structure and thus, a variety
of applications.
What are liposomes?What are liposomes?

3. Liposomes
•invented in 1965 by A. Bangham and from then on they have been
used as a valuable tool in Biology, Biochemistry, Pharmacy and
Therapeutics
IN PHARMACY

’70 –’80
Stealth liposomes ’90’s
Stealth = invisible to the Reticulo-Endothelial system (RES)

4. Liposomes:
“An artificial microscopic vesicle consisting of an aqueous core
enclosed in one or more phospholipid bilayers, used to convey
vaccines, drugs, enzymes, or other substances to target cells or
organs.”
DIAMETER 60nm - 3 microns

5. LIPOSOMES ARE COMPOSED OF NATURAL LIPIDS
(PHOSPHOLIPIDS AND CHOLESTEROL)
LOW RISK OF TOXICITY
cholesterol

8. PhospholipidsPhospholipids

9. LIPOSOME TYPESLIPOSOME TYPES
--ConventionalConventional
--StealthStealth
(with peg molecules on their
surface)
--TargetedTargeted
(with addition of ligands as
antibodies et.c)
--CationicCationic
(with positive surface charge)

11. Preparation of LiposomesSUV are typically 15-30nm in diameter while LUV range from 100-200nm or larger. LUV are stable
on storage, however, SUV will spontaneously fuse when they drop below the phase transition
temperature of the lipid forming the vesicle.

13. Extrusion
 Unilamellar liposomes are
formed by pushing MLV
through polycarbonate
microfilters in extruders, which
results in the narrow
distribution in size of the
liposomal population.
Liposofast Extruder

14. Categories and Naming Size nm Incapsulation Stability
efficiency %
a)Number of lamellae
Unilamellar
Multilamellar (MLV) 500-3000 2 good
b) Size
Small (SUV) 60-100 0.1 medium
Large (LUV) 100-1000 up to 50 good
Giant (GUV) > 1000
c) Preparation technique
Extruded
Detergent removal (DRV)
Reverse evaporation (REV)
CLASSIFICATION

15. Size Determined by Methods
Sonication: SUV
Smaller than 100 nm diameter
Extrusion: LUV (Size depends on the filters)
100 nm—1 µm diameter
Evaporation: GUV
Larger than 1 µm diameter
MLV: Multilamellar vesicles
Monolamellar vesicles:
SUV: Small unilamellar vesicles
LUV: Large unilamellar vesicles
GUV:Giant unilamellar vesicles

16. DSC: differential scanning
calorimetry
Technique that allows to study the phase
transition of lipids around the Melting
Temperature (Tm) by increasing the
temperature of the sample and measuring the
entalpy (∆H).

17. DIFFERENTIAL SCANNING CALORIMETRY (DSC)

21. DRUG ENCAPSULATION

22. Liposome advantagesLiposome advantages
 Retention of both lipophilic and hydrophilic drugs.
 Easy Tailoring, ex. Antibody or ligand conjugation
[targeting]
 Minimum antigenicity.
 Biodegradability
 Biocompatibility

26. Dehydrated-Rehydrated vesicles (DRV)
 Introduced by C. Kirby and G.Gregoriadis, in 1984.
 Empty SUV liposome dispersion is lyophilized (freeze - drying) in presence
of solution of the compound to be entrapped.
 During rehyadration, the addition of small volume of water results in
liposomes with high entrapment efficiency.
 Advantages : simplicity, mild conditions used (important for sensitive
molecules) and high encapsulation efficiency for a variety of compounds.
 Scale-up

27. DRV techniqueDRV technique
Prepare
empty SUV
Mix with
equal volume
of solution of
material to
encapsulate
Freeze
dry until
all water
has been
removed
Rehydrate in a
controlled
Way. Add a very
low volume first
(1/10 of initial)
IMPORTANT: Osmotic pressure of buffers used during rehydration
Rehydration method

28. Other methods
 Detergent removal from mixed lipid-detergent micelles leads
to LUV with large encapsulation volume.
 Freeze Thaw Sonication method (repeated cycles of
liposomes freeze thawing leads to formation of LUV with high
encapsulation efficiency)

29. Purification of drug-entrapping liposomes
Techniques based on size differences of liposomes and
entrapped material:
1. Centrifugation techniques
2. Dialysis
3. “Gel filtration” column chromatography

30. Centrifugation techniques
• This technique is used for large size liposomes: MLV, DRV.
Liposomal
suspension
Centrifugation
15000 rpm for
20 min (25° C)
Add Buffer in
access
Liposomal pellet
(Purification
process is
repeated many
times)
Discard the
supernatant
Add fresh buffer
in access
Resuspend the
liposomal pellet at
the right volume
Purified
liposomal
suspension
Free fluorescence dye
molecules
Encapsulated in liposomes
fluorescence dye

31. Dialysis
• Method used for purification of all types of liposomes
• Sacks of polycarbonate tubing (MW cut off of 10000 Dalton)
• Excess of Buffer solution ( 100 X)
• Dialysis under stirring at 4°C
• Replace the buffer with fresh after 4-5 hours until no fluorescent dye is detected.
Free fluorescence Dye
Access of
Buffer
solution
Encapsulated in liposomes
fluorescence dye
Fig.1. Purification of liposomes by dialysis technique
Free fluorescence dye
molecules
Dialysis
sack

32. Column chromatographic separation
• Sephadex G-50 (polydextran beads) is the material most widely used
for this type of separation To separate free molecules MW<1000
Daltons
Two special points are worth noting with regard to the use
of Sephadex with liposomes:
1. There may be a low yield.
- The problem can be overcome: by making sure that the liposome
sample size is not too small or by pre-saturating the column material
with “empty” liposomes of the same lipid composition as the test
sample )before or after packing the column).

33. 2. Larger liposomes (>0,4μm) may be retained in the column if the
particle size of the gel beads is too small, or if the gel bed contains
too many “fines”.
- The problem can be overcome:
• By Using Medium or coarse grades of Sephadex (particle size 50-
150μm) for chromatography of MLVs (all grades are suitable for
SUVs).

35. Liposomes
Depending upon the site of targeting, liposomes may be coupled
with chemotactic ligands such as peptides, polysaccharides, affinity
ligands like antibodies; pH-sensitive lipids like polyethylenimine or
with hydrophilic PEGylated phospholipids in order to improve their in
vivo performance and to meet a specific therapeutic need.
Date A.A., Adv. Drug Deliv. Rev, 59 2007
Novel systems may incorporate some time-
dependent or other specific inducible changes in
the liposome membrane or its coating to produce
‘intelligent’ liposomes that will change their
properties (e.g. leakage rate, fusogenic activity or
interaction with particular cells) upon a specific
trigger following their application.

36. Filtering (chemical and size exclusion) by the liver and spleen
Barriers to delivery in vivo:
In vivoIn vivo administration ofadministration of LiposomesLiposomes

37. LIPOSOMES ARE ATTACKED BY PLASMA PROTEINSLIPOSOMES ARE ATTACKED BY PLASMA PROTEINS
AFTER IV-INJECTION.AFTER IV-INJECTION.
HDL- Plasma High Density Lipoproteins remove
phospholipid molecules from the vesicle bilayer
Opsonins = Immune and Nonimmune Serum Proteins
which bind to foreign particles and promote phagocytosis.

39. The gel lanes show a size-selective separation of mouse serum proteins in the
corona of the AuNP after incubation and washing. Numbers (kDa) to the left and
right indicate the protein size derived from the marker proteins. Labeled proteins
were detected in corresponding gel bands by MALDI-TOF-MS.

40. =>=> Non-stealth liposomes accumulate in theNon-stealth liposomes accumulate in the liverliver
and spleenand spleen a few minutes after injectiona few minutes after injection
• NATURAL TARGETING (APPLICATIONS IN PARASITIC DISEASES
–leishmaniosis, trypanosomiosis)
• Non-stealth liposomes could not be used to combat other
diseases, due to fast clearance

41. •Small (SUV). more stable.
•Large (LUV). Less stable .
•Negatively charged have a higher tendency to be taken up by the RES than neutral or
positively charged
Filtering (chemical and size exclusion) by the liver and spleen
-Pharmacokinetic Models based on size and charge

42. Sterically stabilized liposomesSterically stabilized liposomes
oror
Stealth liposomesStealth liposomes
 Introduction of
PEG-lipids

43. Liposomes with PEG moleculesLiposomes with PEG molecules
Possible structures KineticsPossible structures Kinetics
«mushroom» conformation
«brush» conformation

45. • There are several liposome formulations that have been commercialized
and there are many liposome formulations that are in various stages of
clinical trials.These are several of the commercialized and phase III
formulations:
• 1) Myocet (Liposomal doxorubicin)- This is a non PEGylated formulation of
liposomal doxorubicin. The liposomes are composed of egg PC (EPC):
cholesterol (55:45 molar ratio). It is used in combinational therapy for
treatment of recurrent breast cancer.
• 2) Doxil, Caelyx (Liposomal doxorubicin)- This is a PEGylated formulation
of liposomal doxorubicin. The liposomes are composed of hydrogenated
soy PC (HSPC): cholesterol: PEG 2000-DSPE (56:39:5 molar ratio). It is used
for treatment of refractory Kaposi's sarcoma, recurrent breast cancer and
ovarian cancer.
• 3) LipoDox (Liposomal doxorubicin)- This is a PEGylated formulation of
liposomal doxorubicin. The liposomes are composed of DSPC: cholesterol:
PEG 2000-DSPE (56:39:5 molar ratio). It is used for treatment of refractory
Kaposi's sarcoma, recurrent breast cancer and ovarian cancer.

46. • 4) Thermodox (Liposomal doxorubicin)- This is a PEGylated formulation of
liposomal doxorubicin. Thermodox is a triggered release formulation. The
liposomes will release their content upon heat. The tumor is heated up
using radio frequency ablation (RFA). The liposomes are composed of
DPPC, mono steroyl PC (MSPC) and PEG2000-DSPE. It is used for
treatment of primary liver cancer (Hepatocellular carcinoma) and also
recurrent chest wall breast cancer. Thermodox is in phase III of clinical
trial.
• 5) DaunoXome (Liposomal Daunorubicin)- This is a non PEGylated
formulation of liposomal Daunorubicin. The liposomes are composed of
DSPC and cholesterol (2:1) molar ratio and it is sized to 45 nm. It is used
for treatment of Kaposi's sarcoma.
• 6) Ambisome (Liposomal Amphotericin B)- This is a non PEGylated
formulation of liposomal Amphotericin B. The liposomes are composed of
HSPC, DSPG, cholesterol and amphoteracin B in 2:0.8:1:0.4 molar ratio. It
is used for treatment of fungal infection.

47. • 7) Marqibo (Liposomal vincristine)- This is a non PEGyated formulation of
liposomal vincristine. The liposomes are composed of egg sphingomylin
and cholesterol. It is used for the treatment of metastatic malignant uveal
melanoma. Marqibo is in phase III of clinical trial.
• 8) Visudyne (Liposomal verteporfin)- This is a non PEGylated formulation
of liposomal verteporfin (BPD-MA). The liposomes are composed of BPD-
MA:EPG:DMPC in 1:05:3:5 molar ratio. It is used for treatment of age-
related macular degeneration, pathologic myopia and ocular
histoplasmosis.

48. • 9) DepoCyt (Liposomal cytarabine)- This is a non PEGylated formulation of
liposomal cytarabine. The Depo-Foam platform is used in DepoCyt. Depo-
Foam is a spherical 20 micron multi-lamellar liposome matrix comprised of
Cholesterol: Triolein: Dioleoylphosphatidylcholine (DOPC):
Dipalmitoylphosphatidylglycerol (DPPG) in 11:1:7:1 molar ratio. The drug is
used by intrathecal administration for treatment of neoplastic meningitis and
lymphomatous meningitis.
• 10) DepoDur (Liposomal morphine sulfate)- This is a non PEGylated
formulation of liposomal cytarabine. The Depo-Foam platform is used in
DepoCyt. Depo-Foam is a spherical 20 micron multi-lamellar liposome matrix
comprised of Cholesterol: Triolein: Dioleoylphosphatidylcholine (DOPC):
Dipalmitoylphosphatidylglycerol (DPPG) in 11:1:7:1 molar ratio. The drug is
used by epidural administration for treatment of postoperative pain following
major surgery.
• 11) Arikace (Liposomal amikacin)- This is a non PEGylated formulation of
liposomal amikacin. The liposomes are composed of DPPC and cholesterol. The
size of the liposomes is between 200-300 nm. It is used for treatment of lung
infections due to susceptible pathogens. Arikace is used in nebulized form and
it is inhaled by the patients. The drug is in phase III of clinical trial.

49. 12) Lipoplatin (Liposomal cisplatin)- This is a PEGylated formulation of
liposomal cisplatin. The liposomes are composed of DPPG, Soy PC,
cholesterol and PEG2000-DSPE. It is used for treatment of epithelial
malignancies such as lung, head and neck, ovarian, bladder and
testicular cancers.
13) LEP-ETU (Liposomal Paclitaxel)- This is a non PEGylated formulation
of liposomal Paclitaxel. The liposomes are composed of DOPE,
cholesterol and cardiolipin. Its is used for treatment of ovarian, breast and
lung cancer. LEP-ETU is completing phase II of clinical trials.
14) Epaxal (Hepatitis A vaccine)- Liposomes have been used as a
vaccine adjuvant in this formulation. These liposomes also known as
immunopotentiating reconstituted influenza virosomes (IRIV) are
composed of DOPC/DOPE in 75:25 molar ratio. The liposomes are sized
to 150 nm.

51. Solid Lipid Nanoparticles (SLN)

52. SLN:SLN:
SOLID LIPID NANOPARTICLESSOLID LIPID NANOPARTICLES
SLN are nanoparticles where the lipid component is composed of
solid lipids (glycerides or waxes) with high Melting point that are
stabilized by using surfactants. SLN are solid at 37°C.
phospholipids,triglicerides, di-glicerides, fatty acids,
cholesterol and cholesterol-ester

53. TRYGLICERIDE
Cetyl- Palmitate (a wax)

54. SLN advantages

55. Method of preparationMethod of preparation::
• High pressure homogenization:
Hot homogenization
Cold homogenization
• Ultrasonication /high speed homogenization:
• Solvent emulsification/evaporation
• Micro emulsion based SLN preparations
• SLN preparation by using supercritical fluid
• Spray drying method
55

56. Hot homogenization
Melting of the lipid & dissolving/dispersing of the drug in the lipid
Dispersing of the drug loaded lipid in a hot aqueous surfactant mixture.
Premix using a stirrer to form a coarse preemulsion
High pressure homogenization at a temperature above the lipid M.P.
Hot O/W nanoemulsion
Solid Lipid Nanoparticles
Disadvantages: 1) temperature induce drug degradation
2) partioning effect
3) complexity of the crystallization
56

58. Ultrasonication/ high speed homogenization :
• SLN were also developed by sonication
• Adv. :
1) Equipment used is very common
2) No temperature induced drug degradation
• Disadv.:
1) Potential metal contamination
2) Broader particle size distribution ranging
into micrometer range.
58

60. DRUG ENTRAPMENT

61. APPLICATIONS
• Solid lipid Nanoparticles possess a better stability and ease of
upscaling as compared to liposomes.
• SLNs form the basis of colloidal drug delivery systems, which
are biodegradable and capable of being stored for at least one
year .
61

62. SLN AS COSMECEUTICALS
• Applied in the preparation of sunscreens.
• SLN have UV reflecting properties.
ORAL SLN IN ANTITUBERCULAR THERAPY
• Anti-tubercular drugs such as rifampicin, isoniazide,
loaded SLNs able to decrease dosing frequency and increase
bioavailability.
SLN AS A GENE VECTOR CARRIER
• Several recent reports of SLN carrying genetic materials such as
DNA, plasmid DNA, & other nucleic acid have been reported.
62

63. • Lipid nanoparticles of reversed internal phase structures, such
as cubic micellar (I2) structure show good drug loading ability
of peptides and proteins as well as some small molecules.
Due to their controllable small size and inner morphology,
such nanoparticles are suitable for drug delivery using several
different administration routes, including intravenous,
intramuscular, and subcutaneous injection
phosphatidylcholine (PC)/glycerol dioleate (GDO)
LIPID MICELLES

64. Lipoplexes
Lipoplexes are complexes of genes (DNA) with cationic lipids and used for
gene therapy
Endocytosis is the major route by which cells uptake NP. Endosomes are
formed as the results of endocytosis, However, if genes can not be released
into cytoplasm by breaking the membrane of endosome, they will be sent to
lysosomes where all DNA will be destroyed before they could achieve their
functions.