Michniak 5: Liposomal Drug delivery

physical chemical facts

- all systems want to reach an equilibrium state of lowest energy

- surface area must be reduced to achieve equilibrium

- hydrophilic molecules always prefer hydrophilic environment; lipophilic prefers lipophilic

interfacial tension

- the force per unit length existing at an interface between 2 immiscible liquids

- one liquid with air on the top-surface tension

- to move a molecule from the inner layers to the surface, work needs to be done against the force of surface tenion

- in other words, each molecule near the surface of a liquid possesses a certain excess of potential energy as compared to the molecules in the bulk of the liquid

to increase the surface area without any additional changes in liquid state:

- work must be done against surface tension without changing liquid temperature

- for a finite change, W = yΔA

- w is work done [surface free energy in ergs]

- y is surface tension in dynes/cm

- ΔA is increase in area in cm^2

liposome

- structure composed of lipid bilayers that encloses volume of aqueous liquid[buffer]

- used as a drug carrier as well as model for biological membranes

- previously a similar approach was used with micelles

- liposomes first prepared by Bangham in 1965 [Cambridge, UK]

types and sizes of liposomes

- small unilamellar vesicles SUVs 0.025-0.05mcm

- large unilamellar vesicles LUVs 0.1 mcm

- multilamellar vesicles MLVs 0.05-10 mcm

- sizes vary with components used, storage, method of prep, type of liposome preparation

materials used for liposomes

- phospholipids: phosphatidylcholine

- steroids

- imparting charge to liposomes: Stearylamine for +ve, dicetyl phosphate for -ve

Important parameters

- captured volume[internal trapped volume]

- encapsulation efficieny

captured volume (internal trapped volume)

- volume enclosed by given amount og lipid with units of L per mole of total lipid

encapsulation efficiency

- fraction of drug sequestered by liposomes

- MLVs better than SUVs for example

advantages of liposomes

- drug carrier made of natural materials, so it doesn't elicit immune response in the body

- carrier which can encapsulate many drugs [hydrophilic and lipophilic]

- can vary size by method of preparation

- can target to RES as well as other areas of the mody [monoclonals]

- carrier protects drug against degradation

- liposomes are degraded to non-toxic by-products

- small enough to deliver IV

disadvantages of liposomes

- destroyed by RES and targeting sometimes difficult to other areas of body

- preparation and reproducibility a challenge

- liposomes tend to be leaky with small MW hydrophilic drugs

- components of liposome liable to peroxidation [place under nitrogen], hydrolysis and oxidation

- can add antioxidants [alfa-tocopherol], freeze dry for reconstitution

methods of liposome preparation

- multilamellar vesicle formation

- sonication for small unilamellar vesicles

- SUVs by extrusion

- SUVs using detergent dialysis

preparation of MLVs

- phospholipid dissolved in chloroform + EtOH-rotary evaporation

- white film of large surface area

- aqueous buffer added-hydration stage

- MLVs form spontaneously-large range of sizes

- sonicate or extrude to get SUVs

SUVs using dialysis

- gives more control over size

- detergents such as bile salts used [Na cholate, Na deoxycholate] and Triton X-100 to solubilize

- after sonification with detergent, place in equipment and dialyze for 12-24 hours

- suspension goes from milky to clear as micelles turn to liposomes

passive encapsulation of hydrophilic and hydrophobic drugs

- hydrophilic requires swelling and separation

- hydrophobic only requires swelling

techniques for physical characterization

-size: electron microscopy, light scattering, ultracentrifugation

- number of lamellae: NMR, small-angle X-ray scattering

- bilayer fluidity: fluorescent probes

- charge: microelectrophoresis

- encapsulated volume: encapsulation of water-soluble markers

different electron micrographs of LUVs

- negative staining

- thin section

- freeze fracture

Amphoterecin Case study

- introduced in 1950s, still broadest antifungal available and little resistance

- natural product of Streptomyces nodosus & is a macrolide polyene antibiotic with length of phospholipid molecule

- Molecule is amphipathic: polyene HC chain is hydrophobic; polyhydroxyl chain is hydrophilic

Amphoterecin important points

- often higher dose is needed to control therapy of immunocompromised patients

- ex: AIDs and transplantation [bone marrow]

- issues of toxicity with these patients, but drug is still best one in the clinic

Amphoterecin B in clinical use

- since 1960

- a polyene macrolide

- isolated from Streptomyces nodosus

- insoluble in water

- solubilized by sodium deoxycholate

- available for IV use as amphotericin B deoxycholate [Fungizone]

lipid amphotericin B formulations

- Abelcet ABLC [amphotericin B lipid complex]

- Amphotec ABCD [amphotericin B colloidal dispersion]

- Ambisome L-AMB [liposomal amphotericin B]

fungizone

- ampho as deoxycholate. Final product is colloidal dispersion of micelles. All negatively charged

Amphotec ABCD

- ampho +Na cholesteryl sulfate; disk shaped particles 122 nm

- negatively charged

Ambisome L-AMB

- al SUVs 50-100 nm, negative charge, lyophilized product

- cannot reconstitute with saline, electrolytes, so you need water or dextrose

Abelcet ABLC

- not liposomes

- ribbon-like lipid particles: 2-5 mcm contain DMPC and DMPG-lipid complex forms

- lowest toxicity and very expensive

stealth liposomes

- special formulation to increase half life of liposomes

- doxorubicin HCl [Doxil] for IV formulated this way

- pegylated liposomes

pegylated liposomes

- binding of methoxy polyethylene glycol [PEG] on surface of liposomes,

- carriers are 100 nm diameter

- circulate undetected by mononuclear phagocyte system in body

- half life prolonged to about 55 hours in circulation

what makes up stealth liposomes

- polymerization of lipids

- polycondensation

- membrane spanning lipids

- insertion of hydrophobic anchor groups

- polymerizable counterions

nystatin

- polyene derived from Streptomyces noursei

- topical antifungal agent

- toxic when administered systemically

- Lisposomal nystatin is a systemic formulation

liposomal nystatin

- may be used for salvage therapy

- in vivo efficacy against candidiasis and aspergillosis

- a potential salvage agent in cases which do not respond to currently available antifungal drugs

Lipid choleates

- first described by Papahadjopoulos in 1975

- obtained as a precipitate after the addition of calcium cations to negatively charged preformed liposomes

- formed mainly out of phosphatidylserine [PS] and calcium

- calcium causes liposomes made from PS to aggregate and fuse

- then Ca brings the bilayers together through partial dehydration and cross-linking of opposing molecules of PS

- success in entrapping amphotericin B

- these systems are stable for ORAL delivery of drugs too

cholate delivery vehicles

- discovered by Papahadjopoulos in 1975

- stable, phospholipid calcium precipitates

- composed of naturally occuring materials

- multilayered structure, containing little or no interal aqueous space

- resistant to degradation in GI tract

- increases shelf life stability

- very inexpensive

process for making choleates

1. calcium interaction with negatively charged lipid displaces H2O and condenses liquid

2. calcium sheets roll up into choleate structures, to minimize contact with water, excluding H2O and O2

membrane fusion of nanocholeates: delivery of siRNA

- Perturbation and reordering of the cell membrane is induced, resulting in a fusion event between the outer layer of the cochleate and the cell membrane.

- The fusion is induced by the formation of Ca(PS)2 complex and results in the delivery of small amount of siRNA into the cytoplasm of the target cell.

- The cochleate could then break free of the cell and be available for another fusion event, either with this or another cell.

- Alternatively, with phagocytic cells, the cochleate may be taken up by endocytosis and the siRNA leach out from it within the endocytic vesicle

stability of choleates

- very stable

- Cochleates can be stored in buffer at 4oC or lyophilized to a powder

and stored at room temperature without altering the structure and the intactness of the encochleated molecules.

in vivo efficacy of Amphotericin B choleates delivered orally

- CAMB (BioDelivery Sciences Inc.) is compared to two commercially

available Amphotericin B preparations, DAMB (desoxycholate amphotericin B, Fungizone; Bristol-Myers Squibb) and LAMB (liposomal amphotericin B, AMBisome; NeXstar Pharmaceuticals)

- oral CAMB at 0.5 mg/kg/day = IP DAMB at 2mg/kg/day, and were superior to LAMB at 10 mg/kg/day

- LAMB showed 10% morality, which is bad

nanoparticles in drug delivery

- metal-based nanoparticles: metal used to anchor drugs

- lipid based nanoparticles: polar groups attached to a hydrophobic tail

- polymer based nanoparticles: amphiphilic copolymers

- biological nanoparticles: viruses

assembly of naoparticles

- drug and the molecules self-assemble into a nano-particle under proper conditions in a liquid medium

challenges of nanoparticles in drug delivery

- stabilization

- extended circulation

- targeting

drug delivery using polymeric micelles

- micelle carrying drug molecules in its core. Polycation-DNA particle in the morphology of a micelle

- endocytosis and transduction deliver the micelle-carried drug into the cell

Topical Paclitaxel Gel Phase 1

- Angiotech announces psoriasis gel determined to be safe and well-tolerated when applied once or twice daily to either normal or psoriatic skin

- problems: toxicity and very low aqueous solubility

Tyrospheres

-tyrosine-derived nanospheres

- used in Paclitaxel PTX to make it 5000X more soluble

- increased solubility creates larger driving force for skin permeation

skin distribution analysis

- fluorescence visualization

- skin sectioning

Tyrospheres in skin distribution of fluorescent dye

- deliver payload to the basal layer of the epidermis but not into the dermis

- disease-state biopsy skin may be a better model for delivery of actives

quantification of delivered Paclitaxel by LC-MS

- skin distribution analysis utilizing cadaver skin show that TyroSpheres deliver significant amounts of drug into the epidermis

- minimal drug found in dermis and receptor fluid->topical delivery

main outcome of TyroSpheres in Paclitaxel Gel

- problems of low solubility and toxicity solved