Targeted Drug Delivery

System

NANOPARTICLES &

LIPOSOMES.

Presented By:

Mr. Nikhil Patil.

M.Pharm 1st year.

Department Pharmaceutics

Introduction

Nanoparticles are sub-nanosized colloidal structurescomposed of synthetic or semi synthetic polymers.

The first reported nanoparticles were based on non-biodegradable polymeric systems.

e.g. polyacrylamide,

polymethylmethacrylate,

polystyrene etc.

The possibilities of chronic toxicity due to tissue and

immunological response towards these polymers had

restricted their use for systemic administration.

This problem has been solved by using biodegradable

polymers.

The term particulate is suggestively generalized because

they could be

Nanospheres

Nanocapsules

Nanocrystals

Nanoparticles

Nanoparticles

Nanospheres Nanoencapsules

Solid core spherical

particle , in which drug

embedded within

matrix or adsorbed on

the surface .

Drug is encapsulated

Within central

volume surrounded

by embryonic

polymeric sheath

Nanospheres and Nanocapsules

Natural Hydrophilic Polymers

• Proteins and Polysaccharides have been

extensively studied and characterized.

Proteins Polysaccharides

Gelatin

Albumin

Lectins

Legumin

Viciline

Alginate

Dextran

Chitosan

Agarsoe

Pullulan

Disadvantage:

1. Batch to batch variation.

2. Conditional biodegradability.

3. Antigenicity.

Synthetic Hydrophobic Polymer :

• The polymer used are either pre-polymerized orpolymerized in process

Pre-polymerized Polymerized in process

Poly (ε - caprolactone)

(PECL)

Poly (lactic acid) (PLA)

Poly ( lactide -co-

glycolide) (PLGA)

Polystyrene

Poly (isobutylcynoacrylates)

(PICA)

Poly (butylcynoacrylates)

(PBCA)

Polyhexylcyanoacrylates

(PHCA)

Poly (methacrylate) (PMMA)

Preparation Techniques

The appropriate method selection depends on the

physicochemical characteristics of the polymer and

the drug to be loaded.

The preparation technique largely determine the

Inner structure

In vitro release profile

Biological fate of the systems.

Preparation Techniques of

Nanoparticles

1) Amphiphilic macromolecule cross linking

a) Heat cross linking.

b) Chemical cross linking.

2) Polymeriazation based method.

a) Polymerization of monomers in situ.

b) Emulsion (micellar) polymerization .

c) Dispersion polymerization.

d) Interfacial condensation polymerization.

d) Interfacial complexation.

3) Polymer precipitation methods

a) Solvent extraction/evaporation

b) Solvent displacement (Nanoprecipitation)

c) Salting out

1. Nanoparticles Preparation by Cross-linking

of Amphiphilic Macromolecules:

Proteins and polysaccharides are used.

This technique involves two steps:

a) The aggregation of amphiphile(s)

b) Stabilization either by heat denaturation or

chemical cross-linking.

These processes may occur in a biphasic o/w or w/o

type dispersed systems, which subdivide the

amphiphile(s) prior to aggregative stabilization.

It may also take place in an aqueous amphiphilic

solution where on removal of the solvent by

extraction or diffusion, amphiphile(s) are aggregated

as tiny particles and subsequently rigidized via

chemical cross-linking.

Cross-linking in w/o emulsion:

Factors governing the size and shape of thenanoparticles are mainly,

- emulsification energy

- temperature

Alternative to this is the chemical cross-linking

method.

Most widely used cross-linking agent is

glutaraldehyde as 3% v/v solution.

The problem associated with the use of chemical as a

cross-linking agent is the complete removal of the

agent.

Emulsion chemical dehydration :

• Hydroxypropyl cellulose solution in chloroform was

used as a continuous phase.

• 2,2, di-methyl propane (Dehydrating agent) was used

to translate internal aqueous phase in to a solid

particulate dispersion.

• produce nanoparticles of size ( 300 nm )

Phase separation in aquious medium

(Desolvation)

The protein or polysaccharide from an aqueous

environment can be desolvated by pH change ,

temperature or by adding appropriate counter ions .

Cross linking may be affected simultaneously or

subsequent to desolvation technique .

This proceeds via three steps

Protein dissolution , protein aggregation and protein

deaggregation

Here sodium sulphate is used as desolvating agent

While Alcohol (ethanol and isopropyl alcohol) are

added as desolvating or deaggregating agent .

Both lipophilic and hydrophillic drugs could be

entrapped in nanoparticles by this method.

pH Induced Aggregation

Here protein phase may be seperated by change of pH.

E.g.-Insulin nanoparticles

Insulin precipitated redissolved

nanodroplet hardened using glutaraldehyde .

Eg- Gelatin & Tween 20 were dissolved in aqueous phase.

pH was adjusted to optimum value .

Clear solution so obtained was heated to 40 0c & followed

by quenching at 40 0c for 24 hours & subsequently left at

ambient temperature for 48 hours .

This lead to gelatin colloidal dispersion .

Finally colloidal aggregate were cross linked using

glutaraldehyde .

Counter Ion Induced aggregation

Protein phase is separated due to presence of counter ions

in aqueous phase.

Aggregation of dispersed phase ( polysaccharide) can be

effectively . Initiated by adding appropriate counter ions.

Aggregation can be propagated by adding secondary

specious of counter ions followed by rigidisation step.

Eg – Alginate nanoparticles

Ca+2 - Gelation inducing agent.

Poly ( L lysine )- Propagation of reaction .

2. Nanoparticle-Preparation Using

Polymerization Based Methods:

a. Polymerization of monomers in situ:

Poly acrylate derivatives are used as polymers.

Two different approaches are generally adopted

for the preparation of nanospheres using this

technique;

i) Emulsion polymerization:

The monomer to be polymerized is emulsified in

a non-solvent phase.

ii) Dispersion polymerization:

The monomer is dissolved in a solvent that is non- solvent

for the resulting polymer.

In emulsion polymerization method, the monomer is

dissolved in an internal phase while in the case of dispersion

polymerization, it is taken in the dispersed phase.

In either of the cases, following polymerization, the polymer

tends to be insoluble in the internal phase or dispersed phase

thus results into an ordered suspension of nanospheres

Micellar Polymerization Mechanism:

Homogenous Polymerization Mechanism:

b) Dispersion polymerization

Monomer is dissolved in aqueous medium , which

act as a precipitant ,for subsequently formed polymer.

Polymerization based method involve in situ

polymerization method where drug to be loaded is

added to formed polymeric nanoparticles .

c. Interfacial Polymerization:

d. Interfacial Complexation:

3. Polymer precipitation methods:

a. Solvent extraction/evaporation

Solvent Evaporation method

b. Solvent displacement (nanoprecipitation)

c. Salting out

Novel Nanoparticulate System

Solid Lipid Nanoparticles.

• These are colloidal carriers (50-100 nm ) which are

composed of physiological lipid dispersed in water or

in an aqueous surfactant solution.

Advantages of SLN :

• Small size and relatively narrow size distribution which

provide biological opportunities for site specific drug

delivery by SLN

• Controlled release of active drug over a long period can

be achieved

• Protection of incorporated drug against chemical

degradation.

• No toxic metabolites are produced.

• Relatively cheaper and stable.

• Ease of industrial scale production by hot dispersion

technique.

Preparation methods of SLN

• Hot Homogenization Technique :

Homogenization of melted lipids at elevated

temperature

• Cold Homogenization Technique :

Homogenization of a suspension of solid lipid at

room temperature

Melting of the lipid

Dissolution of the drug in the melted lipid

Mixing of the preheated dispersion

medium and the drug lipid melt

Hot Homogenization Technique :

High pressure homogenization at a temperature

above the lipids melting point

O/W – nano emulsion

Solidification of the nano emulsion by cooling

down to room temperature to form SLN

Premix using stirrer to form

coarse pre emulsion

Melting of the lipid

• Cold Homogenization Technique :

Dissolution / solubalization of

the drug in the melted lipid

Solidification of the drug loaded lipid in

liquid nitrogen or dry ice

Grinding in a powder mill

(50 – 100 µm particles )

Dispersion of the lipid in the cold

aqueous dispersion medium

Solid Lipid Nanoparticles

Nanocrystals :

Drug

Dispersion with agitation

Surfactant solution

Milling for few hours/day

Nanocrystals

Nanosuspension :

Drug

Dispersion with high speed stirring

Surfactant solution

High pressure homognization 1500 bar pressure

Nano – suspension

Pharmaceutical aspects of

Nanoparticles

• Should be free from potential toxic impurities

• Should be easy to store and administer

• Should be sterile if parentral use is advocated

• Process parameters are performed before releasingthem for clinical trials;

Purification

Freeze drying

Sterilization

Purification of nanoparticles :

Gel filtration :

Remark :

High molecular weight

substances and impurities are

difficult to remove

Purification of nanoparticles :

Dialysis :

Remark :

• High molecular weight

impurities are difficult to

remove

•Time consuming process

Purification of Nanoparticles :

Ultra-centrifugation :

Remark :

• Aggregation of particles

•Time consuming process

Purification of Nanoparticles :

Cross-flow filtration technique:

Freeze drying of Nanoparticles

• This technique involves the freezing of the nanoparticlesuspension and subsequent sublimation of its watercontent under reduced pressure to get free flowingpowder material.

Advantages :

• Prevention from degradation.

• Prevention from drug leakage, drug desorption .

• Easy to handle and store and helps in long termpreservation.

• Readily dispersed in water without modifications intheir physicochemical properties

Sterilization of Nanoparticles :

• Nanoparticles intended for parenteral use should besterilized to be pyrogen free .

• Sterilization can achieved by

Using aseptic technique throughout their preparation,processing and formulation.

Subsequent sterilizing treatments like autoclaving,irradiation.

Characterization of nanoparticles

Parameter Characterization method

Particle size and size distribution

Charge determination Laser Doppler Anemometry

Zeta potentiometer

Chemical analysis of surfaceStatic secondary ion mass spectrometry

Sorptometer

Carrier drug interaction Differential scanning calorimetry

photon correlation spectroscopy

Laser diffractometry

Transmission electron microscopy

Scanning electron microscopy

Atomic force microscopy

Drug stabilityBioassay of drug extracted from nanoparticles

Chemical analysis of drug

Therapeutic application of

nanoparticles

A. Cancer therapy :

• Material –

poly ( alkylcyanoacrylate ) nanoparticles with

anticancer agents, oligonucleotides

• Purpose –

Targeting, reduced toxicity, enhanced uptake of

antitumour agents, improved in vitro and in vivo

stability.

b) Intracellular targeting

• Material :

Poly ( alkylcyanoacrylate ) polyester nanoparticles

with anti-parasitic or antiviral agents

• Purpose :

Targeting reticuloendothelial system for intracellular

infections

c) Prolonged systemic circulation :

• Material :

Polyesters with adsorbed polyethylene glycols or

pluronics or derivatized polyesters

• Purpose :

Prolong systemic drug effect, avoid uptake by the

reticuloendothelial system

d) Occular delivery :

• Material :

poly (alkylcyanoacrylate) nanoparticles with steroids,

anti-inflammatory agents, anti bacterial agents for

glucoma

• Purpose :

improved retention of drug / reduced wash out.

e) DNA delivery :

• Material :

DNA-gelatin nanoparticles, DNA-chitosan

nanoparticles, PDNA-poly(D,L) lactic acid

nanoparticles

• Purpose :

Enhanced delivery and significantly higher expression

levels.

Other applications:

• Poly (alkylcyanocrylate)

nanoparticles with peptides

• Poly (alkylcyanocrylate)

nanoparticles for

transdermal application

• Nanoparticles with a

adsorbed enzymes

• Nanoparticles with

radioactive or contrast

agents

Crosses blood- brain

barrier

Improved adsorption

and permeation

Enzyme

immunoassays

Radio-imaging

Brand name Description Advantages

Emend

(Merck & Co. Inc.)

Nanocrystal aprepiant

(antiemetic) in a capsule

Enhanced dissolution rate

& bioavailability

Rapamune

(Wyeth-Ayerst

Laboratories)

Nanocrystallied Rapamycin

(immunosuppressant) in a

tablet

Enhanced dissolution

rate& bioavailability

Abraxane

(American

Biosciences, Inc.)

Paclitaxel (anticancer drug)

bound albumin particles

Enhance dose tolerance

and hence effect

elimination of solvent

associated toxicity

Rexin-G

(Epeius

Biotechnology

corporation)

A retroviral vector carrying

cytotoxic gene

Effective in pancreatic

cancer treatment

Targeted Drug Delivery System

LIPOSOMES

What are Liposomes?

• They are simply vesicles or ‘bags’ in which an

aqueous volume is entirely enclosed by a membrane

composed of lipid (fat) molecules, usually

phospholipids.

These vesicles can encapsulate water-soluble drugs in

their aqueous spaces and lipid soluble drug within

the membrane itself.

• Structurally, liposomes are bilayered vesicles in

which an aqueous volume is entirely enclosed by a

membranous lipid bilayer mainly composed

of natural or synthetic phospholipids.

Advantages of liposome :

• Provides selective passive targeting to tumor tissues

• Increased efficacy and therapeutic index

• Increased stability via encapsulation

• Reduction in toxicity of the encapsulated agent.

• Improved pharmacokinetic effects

• Used as carriers for controlled and sustained drug

delivery

• Can be made into variety of sizes.

Disadvantages of liposome :

• Leakage of encapsulated drug during storage.

• Uptake of liposomes by the reticuloendothelial system

• Batch to batch variation

• Difficult in large scale manufacturing and sterilization

• Once administered, liposomes can not be removed

• Possibility of dumping, due to faulty administration

Mechanism of liposome formation• In order to understand why liposomes are formed when

phospholipids are hydrated, it requires a basic

understanding of physiochemical features of

phospholipids.

• Phospholipids are amphipathic molecules (having affinity

for both aqueous and polar moieties) as they have a

hydrophobic tail is composed of two fatty acids

containing 10-24 carbon atoms and 0-6 double bonds in

each chain.

• In aqueous medium the phospholipids molecules are

oriented in such a way that the polar portion of the

molecule remains in contact with the polar environment

and at the same shields the non-polar part.

• They align themselves closely in planer bilayer sheets to

minimize the interaction between the bulky aqueous

phase and long hydrocarbon fatty acyl chains.

• This alignment requires input of sufficient amount of

energy (in the form of shaking, sonication,

homogenization, heating, etc).

• Interactions are completely eliminated when these

sheets fold over themselves to form closed, sealed

and continuous bilayer vesicles.

Classification of liposome's

1) Based on structural parameters

MLV, OLV,UV,SUV,MUV,LUV,GUV,MV.

2) Based on method of liposome preparation

REV, MLV-REV, SPLV, FATMLV, VET, DRV.

3) Based on the composition and application

CL, RSVE, LCL ,pH sensitive liposome, cationic

liposome , immuno- liposomes .

Materials used in preparation of

liposomes

A) Phospholipids :

• It is the major component of the biological membrane.

• Two types of phospholipids are used natural and syntheticphospholipids.

• The most common natural phospholipid is the phospatidylcholine(PC) is the amphipathic molecule and also known as lecithin.

• It is originated from animal (hen egg) and vegetable (soya bean).

B. Steroids :

• Cholesterol is generally used steroid in the formulation

of liposomes.

• It improves the fluidity of the bilayer membrane and

reduces the permeability of bilayer membrane in the

presence of biological fluids such as blood / plasma.

• Cholesterol appears to reduce the interactions with

blood proteins.

Methods of liposomes

preparations

Passive loading technique

Active loading

technique

Mechanical dispersionmethods

Solvent dispersion methods

Detergent removal methods

Mechanical dispersion methods

Lipid is solublised in organic solvent, drug to be

entrapped is solubilise in aqueous solvent, the lipid phase

is hydrated at high speed stirring due to affinity of aqueous

phase to polar head it is entrapped in lipid vesicles.

e.g. Lipid film hydration, Micro-emulsification.

(Micro fluidizer ), Sonication.

Solvent dispersion methods

In this method, lipids are first dissolved in organic

solvent, which then brought in to contact with

aqueous phase containing material which is to be

entrapped in liposome under rapid dilution and rapid

evaporation of organic solvent.

E.g. Ethanol injection

Ether injection

De-emulsification

Detergent removal method

In this methods, the phospholipids are brought into

intimate contact with the aqueous phase via detergent

which associate with phospholipids molecules and serve to

screen the hydrophobic portions of the molecules from

water.

Detergent (Cholate, Alkyglycolate, Triton X-100)

removal from mixed micells by

Dialysis

Column chromatography

Dilution

Surface charge Free-flow electrophoresis

Electrical surface potential and surface pH

Zeta potential measurements & pH sensitive probes

Percent of free drug/ percent capture

Drug release Diffusion cell/ dialysis

Parameter Characterization method

Vesicle shape and surface morphology

Mean vesicle size and size distribution

Dynamic light scattering, zetasizer, Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion

Mini column centrifugation, ion-exchange chromatography, radiolabelling

Transmission electron microscopy, Freeze-fracture electron microscopy

Physical Characterization

Phopholipid peroxidation UV absorbance, Iodometric and GLC

Phospholipid hydrolysis, Cholesterol auto-oxidation

HPLC and TLC

Osmolarity

Parameter Characterization method

Phospholipid concentration

Cholesterol concentration Cholesterol oxidase assay and HPLC

Osmometer

Barlett assay, stewart assay, HPLC

Chemical Characterization

Animal toxicity Monitoring survival rates, histology and pathology

Parameter Characterization method

Sterility

Pyrogenicity Limulus Amebocyte Lysate (LAL) test

Aerobic or anaerobic cultures

Biological Characterization

Stability

• Physical stability :

Once liposome are formed, they behave similar to the

other colloidal particles suspended in water.

Neutral particles tend to aggregate or flocculate and

sediment with increase in size on storage. Adding

charged lipids such as stearyl amine, diactyl phosphate

and phosphatidyl serine can control the aggregation.

The addition of charged lipids causes repulsion and

prevents major changes in the overall size of liposome.

• Chemical stability :

Phospholipids, especially those derived from natural

sources, are subject to two major degradative reaction

A. Lipid peroxidation : most phospolipid liposomes

contain unsaturated acyl chains as part of their

molecular structure and susceptible to oxidative

degradation. It can be minimized by the use of animal

derived lipids like egg PC, which has less saturated

lipids, use of light resistant containers, use of

antioxidants are useful in minimizing oxidation.

B. Lipid hydrolysis :

hydrolysis in phospholipids results in the formation of

free fatty acids and lyso-lecithin. Selecting a good source

of lipid, temperature, pH, and minimizing oxidation.

• Biological stability :

liposome's release entrapped molecules rapidly when

incubated with blood or plasma. This instability is

attributed to the transfer of bilayer lipids to albumin and

high density liposome.

Therapeutic applications of liposomes1. Liposomes as drug / protein delivery vehicles

• Controlled and sustain release in situ

• Enhanced drug solubilization

• Enzyme replacement therapy and lysosomal storagedisorders

• Altered pharmacokinetics and biodistribution

2. Liposomes in antimicrobial, antifungal and antiviraltherapy

3. Liposomes in tumour therapy

• Carrier of small cytotoxic molecules

• Vehicle for macromolecules as cytokines and genes

4. Liposome in gene delivery

• Genes and antisense therapy

• Genetic (DNA) vaccination

5. Liposome in immunology

6. Liposome as radiopharmaceutical and radio

diagnostic carrier

7. Liposome in cosmetic and dermatology

8. Liposome in enzyme immobilization and bioractor

technology

Drug Route of

administration

Targeted

Diseases

Amphotericin-B Oral delivery Mycotic infection

Insulin Oral, Ocular, Pulmonary

and Transdermal delivery

Diabetic mellitus

Ketoprofen Ocular delivery Pain muscle condition

Pentoxyfylline Pulmonary delivery Asthma

Tobramycin Pulmonary delivery Pseudomonas infection,

aeruginosa

Drug Route of

administration

Targeted Diseases

Salbutamol Pulmonary delivery Asthma

Benzocain Transdermal ulcer on mucous surface with

pain

Ibuprofen Oral delivery Rheumatoid arthritis

Adrenaline Ocular delivery Glucoma, Conjectivitis

Penicillin G Pulmonary delivery Meningococal,

staphylococcal

Methotrexate Transdermal Cancer

Marketed

product

Drug used Target

diseases

Company

DoxilTM or

CaelyxTM

Doxorubicin Kaposi’s sarcoma SEQUUS, USA

DaunoXomeTM Daunorubicin Kaposi’s sarcoma,

breast & lung

cancer

NeXstar, USA

AmphotecTM Amphotericin-B fungal infections,

Leishmaniasis

SEQUUS, USA

VENTUSTM Prostaglandin-E1 Systemic

inflammatory

diseases

The liposome

company, USA

ALECTM Dry protein free

powder of DPPC-

PG

Expanding lung

diseases in babies

Britannia Pharm,

UK

Reference

a) Targeted and controlled drug delivery, S.P.Vyas and

R.K.Khar, CBS Publication 2008,

Nanoparticles – page no 331 to 386.

Liposomes – page no 173 to 248.

b) Controlled And Novel drug delivery – By N.K.Jain.

c) Novel Drug Delivery system by Y.W.Chien

d) Text book of Industrial Pharmacy, Shobha Rani

Hiremath, Orient Longman Private ltd.

e) www.google.com