1. LIPOSOMES
AND
SOLID LIPID NANOPARTICLES

2. What are liposomes?
• 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.

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 layers, used to convey
vaccines, drugs, enzymes, or other substances to target cells or
organs.”
A spherical particle in an aqueous medium, formed by a lipid
bilayer enclosing an aqueous compartment
SIZE 60nm - 3 microns

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

8. Phospholipids

9. LIPOSOME TYPES
-Conventional
-Stealth
(with peg molecules on their
surface)
-Targeted
(with addition of ligands as
antibodies et.c)
-Cationic
(with positive surface charge)

12. Preparation of Liposomes
SUV 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.

14. 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

15. Size Determined by Methods
MLV: Multilamellar vesicles
Monolamellar vesicles:
SUV: Small unilamellar vesicles
LUV: Large unilamellar vesicles
GUV:Giant unilamellar vesicles
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

16. Characterization
• Drug encapsulation (D/L)
• Size distribution (DLS, cryo-TEM, SEM, AFM)
• Zeta-potential (surface charge)
• Stability (size)
• Integrity /retention of drug (calcein
technique)
– In presence of plasma proteins
– In presence of Ca2+

17.  The critical parameters of a nanoparticulate formulation to set and monitor
quality standards have to be based on simplicity (for routine analysis),
reliability and correlation to the in vivo performance.
o Particle size
o Zeta potential
o Polydispersity Index
o pH of the suspension
o Aggregation?
o Redispersibility
o Assay of the incorporated drug
o Maximum allowable limit of solvents
o Residual stabilizer
o Degradation products (oligomers/monomers)
Journal of Biomedical Nanotechnology. 1 (2005) 235-258 17
Nanomedicine: Nanotechnology, Biology and Medicine 2(2006) 127-136

18. 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).

19. DIFFERENTIAL SCANNING CALORIMETRY (DSC)

23. DRUG ENCAPSULATION

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

28. 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

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

30. 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)

31. 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

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

33. 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
Encapsulated in liposomes
Access of fluorescence dye
Buffer Free fluorescence dye
solution molecules
Dialysis
sack
Fig.1. Purification of liposomes by dialysis technique

34. 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).

35. 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).

37. Liposomes
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.
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

38. In vivo administration of Liposomes
Barriers to delivery in vivo:
Filtering (chemical and size exclusion) by the liver and spleen

39. LIPOSOMES ARE ATTACKED BY PLASMA PROTEINS
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.

41. => Non-stealth liposomes accumulate in the liver
and spleen a 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

42. Filtering (chemical and size exclusion) by the liver and spleen
-Pharmacokinetic Models based on size and charge
•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

43. Sterically stabilized liposomes
or
Stealth liposomes
 Introduction of
PEG-lipids

44. Liposomes with PEG molecules
Possible structures Kinetics
«mushroom» conformation
«brush» conformation

46. • 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.

47. • 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.

48. • 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.

49. • 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.

50. 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.

52. Solid Lipid Nanoparticles (SLN)
Lipid utilized ( SLN )
• phospholipids,triglicerides, di-glicerides, fatty acids,
cholesterol and cholesterol-ester

54. SLN:
SOLID LIPID NANOPARTICLES
SLN are nanoparticles where the lipid component is composed of
solid lipids (glycerides or cere) with high Melting point that are
stabilized by using surfactants. SLN are solid at 37°C.

55. SLN advantages

56. Preparation of SLN

57. 70°C
70°C
70°C
25°C