LIPID-BASED NANOPARTICLES:

LIPOSOMES

SOLID LIPID NANOPARTICLES

LIPID MICELLES

LIPOPLEXES

• 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?

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)

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 compartmentDIAMETER 60nm - 3 microns

LIPOSOMES ARE COMPOSED OF NATURAL LIPIDS (PHOSPHOLIPIDS AND CHOLESTEROL)

LOWER RISK OF TOXICITY

cholesterol

PhospholipidsPhospholipids

LIPOSOME TYPESLIPOSOME TYPES

--ConventionalConventional--StealthStealth(with peg molecules on their surface)

--TargetedTargeted(with addition of ligands as antibodies et.c)

--Cationic Cationic (with positive surface charge)

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.

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

Categories and Naming Size nm Incapsulation Stabilityefficiency %

a)Number of lamellaeUnilamellarMultilamellar (MLV) 500-3000 2 good

b) Size Small (SUV) 60-100 0.1 mediumLarge (LUV) 100-1000 up to 50 goodGiant (GUV) > 1000

c) Preparation technique Extruded Detergent removal (DRV) Reverse evaporation (REV)

CLASSIFICATION

Size Determined by Methods

Sonication: SUV Smaller than 100 nm diameterExtrusion: LUV (Size depends on the filters) 100 nm—1 µm diameterEvaporation: GUV Larger than 1 µm diameter

MLV: Multilamellar vesicles

Monolamellar vesicles:SUV: Small unilamellar vesiclesLUV: Large unilamellar vesiclesGUV:Giant unilamellar vesicles

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).

DIFFERENTIAL SCANNING CALORIMETRY (DSC)

DRUG ENCAPSULATION

Liposome advantagesLiposome advantages

Retention of both lipophilic and hydrophilic drugs. Easy Tailoring, ex. Antibody or ligand conjugation

[targeting]

Minimum antigenicity. Biodegradability Biocompatibility

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

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 rehydrationRehydration method

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)

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

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

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

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).

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).

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.

Filtering (chemical and size exclusion) by the liver and spleen

Barriers to delivery in vivo:

In vivo In vivo administration ofadministration of LiposomesLiposomes

LIPOSOMES ARE ATTACKED BY PLASMA PROTEINS LIPOSOMES 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.

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.

=> => Non-stealth liposomes accumulate in the Non-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

•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

Sterically stabilized liposomesSterically stabilized liposomesoror

Stealth liposomesStealth liposomes

Introduction of

PEG-lipids

Liposomes with PEG moleculesLiposomes with PEG molecules

Possible structures KineticsPossible structures Kinetics

«mushroom» conformation

«brush» conformation

• 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.

• 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.

• 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.

• 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.

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.

Solid Lipid Nanoparticles (SLN)

Lipid utilized ( SLN )

• phospholipids,triglicerides, di-glicerides, fatty acids, cholesterol and cholesterol-ester

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.

SLN advantages

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

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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

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Cold homogenization

Melting of lipid & dissolving/dispersing of the drug in the lipid Solidification of the drug loaded lipid in liquid nitrogen or dry ice Grinding in a powder mill Dispersing the powder in a aqueous surfactant dispersion medium High pressure homogenization at room temperature or below. Solid Lipid Nanoparticles

Disadvantages: 1) Larger particle sizes & broader size distribution 2) does not avoid thermal exposure but minimizes it

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Ultrasonication/ high speed homogenization :

• SLN were also developed by high speed stirring or 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.

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DRUG ENTRAPMENT

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 .

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SLNS AS COSMECEUTICALS

• Applied in the preparation of sunscreens.• SLN has 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.

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• 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

LipoplexesLipoplexes 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.