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Liposome Encapsulation
Liposomes are bilayer (double-layer),
liquid-filled bubbles made from
phospholipids. Over 50 years ago,
researchers discovered that these spheres
could be filled with therapeutic agents and
used to protect and deliver these agents
into the body and even into specific cells
of the body.
The therapeutic value and greatly
increased delivery of liposomeencapsulated drugs and nutrients.
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
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Liposome Encapsulation
Lipids, Phospholipids and Liposomes
 Lipids are a group of naturally occurring molecules that include fats,
waxes, sterols, fat-soluble vitamins. The main biological functions of
lipids include storing energy, signaling, and acting as structural
components of cell membranes.
 Phospholipids are a class of lipids that are a major component of all
cell membranes as they can form lipid bilayers. The structure of the
phospholipid molecule generally consists of hydrophobic tails and a
hydrophilic head.
 A liposome is a spherical vesicle having at least one lipid bilayer. The
liposome can be used as a vehicle for administration of nutrients and
pharmaceutical drugs. Liposomes can be prepared by disrupting
biological membrane.
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
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Liposome Encapsulation
Liposomal formulations
 Since the first liposomal pharmaceutical product, Doxil, was approved in
1995 there are now several successful liposomal formulations.
 Most of them have to be administrated intravenously due to the
degradation of lipids in the gastrointestinal tract. However, some recent
formulations such as Arikace can be subcutaneously injected or inhaled as
aerosols.
 Apart from a broadened range of drugs being investigated for liposomal
formulations, new strategies such as environmental sensitivity and
combination therapy have been applied to the development process to
achieve better efficacy. Moreover, liposomes could be successfully applied
to areas other than cancer therapy, such as vaccines.
 The next slide shows a table of clinically approved liposomal formulations.
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
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Liposome Encapsulation
Clinically Approved Liposomal Drugs
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177
Provider of Global Contract Research Services
Accelerating Preclinical Research, Drug Discovery & Therapeutics
Services >
Liposome Encapsulation
 Altogen Labs offers encapsulation of any charged
 Standard formulations
molecuIe such as mRNA, siRNA, shRNA,
including all of the
microRNA, plasmid DNA, protein
 The encapsulated molecule can be transformed
into either one of several standard liposome
formulations such as PC : Cholesterol and
DSPC : Cationic Lipid : PEGylated Lipoid :
Cholesterol or a custom liposome formulation
 This can be done in vitro or in vivo
formulations listed in
previous table
 Custom formulation
 Developing formulation
methods
 Encapsulation of the
compound of interest into
liposomal formulation
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177
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Liposome Encapsulation
Benefits and Applications
 Due to their unique properties, including low cytotoxicity, good
biocompatibility and biodegradability, liposomes possess wide
applications in different fields including gene and drug delivery, food and
nutrition industries and cosmetic industries.
 Recently, a number of methods have been developed in order to modify
the liposome structure and improve their stability as well as establishing
high concentrations of bioactives in the target cells and cellular
compartments in order to gain maximum therapeutic efficiency.
 Active targeting can be achieved via appropriately engineered
modifications to the liposomal structure. For active targeting of liposomes,
thermo-labile, pH-sensitive, photo-sensitive and antibody coated vesicles,
have been designed.
 Through passive targeting, the bioactive-carrier complex reaches its
destination based on the physicochemical properties of bioactive carrier
complexes and does not require utilization of any targeting strategy.
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Liposome Encapsulation
Mechanism
 A liposome has an aqueous solution core surrounded by a hydrophobic
membrane, in the form of a lipid bilayer.
 Hydrophilic solutes dissolved in the core cannot readily pass through the
bilayer. Hydrophobic chemicals associate with the bilayer.
 A liposome can be hence loaded with hydrophobic and/or and hydrophilic
molecules. To deliver the molecules to a site of action, the lipid bilayer can
fuse with other bilayers such as the cell membrane, thus delivering the
liposome contents.
 Useful liposomes rarely form spontaneously. They typically form after
supplying enough energy to a dispersion of (phospho)lipids in a polar
solvent, such as water, to break down multilamellar aggregates into oligoor unilamellar bilayer vesicles.
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
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Liposome Encapsulation
Mechanism
 By preparing liposomes in a solution of DNA or drugs (which would
normally be unable to diffuse through the membrane) they can be
(indiscriminately) delivered past the lipid bilayer, but are then typically
distributed non-homogeneously.
 For drug delivery, liposomes that contain low (or high) pH can be
constructed such that dissolved aqueous drugs will be charged in solution
(i.e., the pH is outside the drug's pH range).
 As the pH naturally neutralizes within the liposome (protons can pass
through some membranes), the drug will also be neutralized, allowing it to
freely pass through a membrane. These liposomes work to deliver drug by
diffusion rather than by direct cell fusion.
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177
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Liposome Encapsulation
Evolution of Liposome
First generation: Conventional
Liposomes
Various types of drugs can be loaded
into the interior, in the bilayer or at the
interface depending on the nature of the
compounds.
Second generation: PEGylated
Liposome
It offers significant advantages over
conventional liposomes such as prolonged
blood circulation time and hence enhanced
drug accumulation at the disease site. Other
potential benefits include reduced toxicity,
reduced dosing, frequency etc.
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Liposome Encapsulation
Evolution of Liposome
Third generation: Ligand-Targeted
Liposome
This is the most advanced liposome
system. PEGylated liposomes with
targeting ligands (antibodies, antibody
fragments, peptides and small
molecules) have the potential of specific
targeted delivery to the disease site
through ligand-receptor binding
Lipid-Nanoparticles
(Liposphere)
Liposphere is a nano-particle
with an lipophilic core (such as
triglycerides) coated with a
monolayer of lipids.
Lipophilic drugs are loaded in the
oil core.
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Liposome Encapsulation
Factors that affect liposome
preparation method
 The physicochemical characteristics of the material encapsulated
and those of the liposomal ingredients
 The nature of the medium in which the lipid vesicles are dispersed
 The effective concentration of the entrapped substance and its
potential toxicity
 Optimum size, polydispersity and shelf-life of the vesicles for the
intended application
 Batch-to-batch reproducibility and possibility of large-scale
production of safe and efficient liposomal products
 Additional processes involved during application/delivery of the
vesicles
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177
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Liposome Encapsulation
Methods of Liposome Preparation
 Ether/alcohol injection
 Freeze dry evaporation method
 Extrusion method
 Reverse phase evaporation
 Microfluidization (Microfluidics)
 Detergent depletion
 Supercritical fluid injection and decompression
 Dense gas techniques
 Dual asymmetric centrifugation
 High-pressure homogenization
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177
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Liposome Encapsulation
Protocols: Freeze-dry
Freeze-drying of liposomes
can prevent hydrolysis of
phospholipids and also help
to stabilize encapsulated
material.
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Liposome Encapsulation
Protocols: Extrusion
 Liposome extrusion is a widely used method in which liposomes are
forced under pressure through filters with defined pore sizes in order to
generate a homogenous population of smaller defined vesicles.
 Prior to extrusion through the final pore size, multilamellar lipsome (LMV)
suspensions are disrupted either by several freeze-thaw cycles or by
prefiltering the suspension through a larger pore size (typically 0.2µm1.0µm). This method helps prevent the membranes from fouling and
improves the homogeneity of the size distribution of the final suspension.
 Extrusion through filters with 100nm pores typically yields large,
unilamellar vesicles (LUV) with a mean diameter of 120-140nm. Mean
particle size also depends on lipid composition and is quite reproducible
from batch to batch.
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177
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Liposome Encapsulation
Protocols: Microfluidization
 A major challenge in the development of liposomes for drug delivery is the control of size
and size distribution. Microfluidics is an emerging technology for liposome synthesis,
because it enables precise control of the lipid hydration process and allows for the
production of liposomes ranging from tens of nanometers to tens of micrometers in
diameter.
 Isopropyl alcohol (IPA) containing the dissolved lipids flows through the center inlet
channel, and an aqueous solution flows through the two side inlet channels. The stream of
lipids in IPA is hydrodynamically focused by two aqueous streams at the cross junction of
the microfluidic chip. The liposome formation is based on a diffusion-driven process in
which the dissolved lipids self-assemble into liposomes as IPA quickly diffuses and dilutes
into two aqueous streams at the interfacial region.
 The lipid IPA solution is injected into the center channel of the microfluidics network, while
phosphate-buffered saline (PBS) is injected into two side channels intersecting with the
center channel. Relatively high liposome concentration can be produced at the center point
in the channel once the focused IPA stream is diluted to the critical concentration for
formation of the more stable liposomes along the interfacial region.
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Liposome Encapsulation
Protocols: DNA plasmid encapsulation
Chloroform solutions of lipids (10–20 mg/ml) are mixed in a 12×75 mm2 glass tube at the following
composition (20 μmol total lipid, mole percent): Cholesterol 55%, DSPC 20%, DDAB 15% and
DSPE-PEG2000 10%. The solvent is evaporated under vortexing and under a thin nitrogen gas
stream allowing a thin, fairly even lipid film to form on approximately 6 cm of the glass surface. High
vacuum is applied overnight to ensure complete solvent evaporation. A Tris–HCl buffer (300 μl,
50 mM, pH 7.0) is used to hydrate the lipids and allow for vesicle formation. The tube is rotated and
lipids allowed to hydrate overnight at room temperature. The next day the liposome preparation is
placed in a basket and sonicated for 2 min using a Bransonic water bath. Plasmid DNA (Endo-free) is
added to the tube and after collecting the material at the bottom of the tube by a brief spin exactly
one volume of 80% ethanol in Tris-buffer (50 mM, pH 7.0) is added dropwise and with mixing during
one minute.
The tube is subjected to five cycles of freeze-thaw between dry ice/EtOH and 37 °C water bath with
2–3 min in each step. Liposomes are downsized using 11 passes in a hand-held, small-scale
extruder with polycarbonate nucleopore filters (400 nm, 200 nm and 100 nm). For each step a small
volume of buffer to wash the extruder ensures a complete liposome recovery. The entire SPLP
volume (typically 1 ml) after the extrusion process is transferred to a dialysis cassette (10 kDa
MWCO) and dialyzed against 0.5 l HEPES buffer (pH 7.4) overnight at room temperature with one
buffer exchange.
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Liposome Encapsulation
Protocols: Protein encapsulation
Preparation of liposomes
Liposomes are prepared by an ethanol injection method. Briefly, varied molar ratios of
DSPC/cholesterol/DSPE-PEG/DHA are melted in a 60°C water bath, and the lipid mixture is then
dissolved in the proper alcohol solvent and rapidly injected at room temperature with a one-way, 1 mL
syringe into a dispersant water solution. The dispersion is extruded ten times through a polycarbonate
membrane (100 nm pore size) using an extruder. The various ratios of lipid content/ethanol/water (L/E/W)
are evaluated.
Preparation of lipoparticles
Solid cores are prepared using a physical mix method. Briefly, FITC-BSA
and protamine sulfate are freshly dissolved in ethanol. The weight ratio
of protamine sulfate/FITC-BSA (20/1) solution is added and mixed. An
FITC-BSA/protamine sulfate core is dissolved in the alcohol lipid
solution; the correct aliquot of this solution is injected as in the liposome
preparation process described above. Untrapped FITC-BSA and
protamine sulfate from the lipoparticles is removed by ultracentrifugation
at 25,000 g for 15 minutes and further dialyzed against 800 mL distilled
water for 12 hours at 4°C to remove untrapped FITC-BSA from the
lipoparticle vesicles.
Provider of Global Contract Research Services
Accelerating Preclinical Research, Drug Discovery & Therapeutics
Endpoints
 Altogen Labs offers encapsulation of any charged
molecuIe such as mRNA, siRNA, shRNA,
microRNA, plasmid DNA, protein
 The encapsulated molecule can be transformed
into either one of several standard liposome
formulations such as PC : Cholesterol and
DSPC : Cationic Lipid : PEGylated Lipoid :
Cholesterol or a custom liposome formulation
 Standard formulations
including all of the
formulations listed in
previous table
 Custom formulation
 Developing formulation
methods
 Encapsulation of the
compound of interest into
liposomal formulation
 This can be done for in vitro or in vivo studies
Altogen Labs • 11200 Manchaca Road • Suite 203 • Austin • TX • 78748 • USA
[email protected] • (512) 433-6177