TDDS, Targeted drug delivery system, Targeted system

1. TargetedDrug Delivery Systems
Presented by;
K. Sai Lakshmi
B.Pharmacy, final year,
(2016-2020)
Balaji College of Pharmacy.

2.  Targeted drug delivery system have been designed on the concept of magic
bullets given by Dr.Paul Ehrlich.
 This concept is associated with the development of such systems which when
introduced in the body, direct the drug only to its site of action therapy providing
maximum therapeutic response with reduced toxic effects due to decreased
distribution of other body tissues.
 Targeted drug delivery system are those in which maximum drug concentration
is achieved at the specific site of drug action either by using inert forms of active
drug or by utilizing specially designed polymers.
 Targeted drug delivery system which employ a biologically inert polymer as a
carrier to carry the drug to its site of action are referred as drug-carrier
delivery systems. In these, the drug can be either entrapped within the carrier
or covalently bonded to it.
 Several promising drug -carrier system have been developed, which utilize
nanoparticles, liposomes, RBCs, niosomes, microcapsules, vesicular
graft etc., as carrier molecules.

3. Ideal properties of Drug- Carrier Delivery Systems
 The Drug-carrier delivery systems should possess the following
characteristics.
 They should enhance the drug action by prolonging its systemic circulation.
 They should increase the drug concentration at its site of action.
 They decrease or prevent tissue toxicity.
 They should prevent the drug from undergoing metabolic degradation by
providing adequate protection.
 They should continue the drug with in the desired body tissue(s).
 They should carry the drug during the transit and release it only at the site
of action at an appropriate rate.

4. Merits of Targeted drug delivery system
 These system decreases the amount of drug required for administration, which is
beneficial for drugs with low therapeutic index.
 Maximum amount of the drug administered reaches the site of action. Therefore
only least quantities reach the other body tissues.
 Enables the drug to elicit maximum therapeutic activity.
 Prevent the degradation or inactivation of drug during as transport to the site of
action.
 Prevent any adverse reaction due to drug induced toxicity.
Demerits of Targeted drug delivery system
 Rapid clearance of targeted system.
 Immune reaction against i.v administration carrier system.
 Diffusion and Redistribution of released drug.
 Drug deposition at the target site may produce toxicity symptoms.
 Difficult to maintain stability of dosage form.

5. LIPOSOMES
 Liposomes are concentric bilayered vesicles in which an aqueous core is entirely
enclosed by a membranous lipid bilayer mainly composed of natural or synthetic
phospholipids.
The size of a liposome ranges from some 20 nm up to several micrometers.
 The lipid molecules are usually phospholipids- amphipathic moieties with a
hydrophilic head group and two hydrophobic tails.
 On addition of excess water, such lipid moieties spontaneously originate to give the
most thermodynamically stable conformation.
 In which polar head groups face outwards into the aqueous medium, and the lipid
chains turns inwards to avoid the water phase, giving rise to double layer or bilayer
lamellar structures.

6. COMPOSITION OF LIPOSOMES
The major components of liposomes are phospholipids and cholesterol

7. PHOSPHOLIPIDS
 Phospholipids are major structural components of biological membranes in
human body, where 2 types of phospholipids exist i.e. phosphodiglycerides &
sphingolipids .
 Each phospholipid molecule has 3 major parts, 1 head & 2 tails. Head is made
from 3 molecular components: choline , phosphate & glycerol which is
hydrophilic. Each tail with a long chain EFA which are hydrophobic.
 Most commonly used phospholipids – PC an amphipathic molecule with a
hydrophilic polar head group, phosphocholine . PC, also known as “lecithin”, can
be derived from natural and synthetic sources.

8.  The lipid bi-layer used in the liposomes are usually made of
phospholipids and cholesterol.
Following are the
A)Naturally occurring phospholipids used in liposomes are:
• Phosphatidylcholine (PC),
• Phosphatidylethanolamine (PE),
• Phosphatidylserine (PS).
B) Synthetic phospholipids used in the liposomes are:
• Dioleoyl phosphatidylcholine (DOPC),
• Distearoyl phosphatidylcholine (DSPC),
• Dioleoyl phosphatidylethanolamine (DOPE),
• Distearoyl phosphatidylethanolamine (DSPE).

9. CHOLESTEROL
 Incorporation of sterols in liposome bilayer can bring about major changes in the
preparation of these membranes.
 Cholesterol does not by itself form bilayer structure , it acts as fluidity buffer.
 It inserts into membrane with hydroxyl group oriented towards aqueous surface
& aliphatic chain aligned parallel to acyl chains in the centre of bilayer.
 It can be incorporated into phospholipid membranes in very high Conc. upto 1:1
or even 2:1 molar ratios of PC.
 Cholesterol incorporation increases the separation between the choline head
groups and eliminates the normal electrostatic and hydrogen-bonding
interactions.

10. Role of cholesterol in bilayer formation:
1. Cholesterol act as fluidity buffer.
2. After intercalation with phospholipid molecules alter the freedom of motion
of carbon molecules in the acyl Chain
3. Restricts the transformations of trans to gauche conformations.

11. Morphology of Liposomes

13. Advantages of liposomes
 Provides selective passive targeting to tumor tissues.
 Increased efficacy and therapeutic index.
 Increased stability of encapsulated drug.
 Reduction in toxicity of the encapsulated agent.
 Site avoidance effect (avoids non-target tissues).
 Improved pharmacokinetic effects (reduced elimination increased circulation life ti
me.
 Flexibility to couple with site specific ligands to achieve active targeting.
 The size, surface charge and other characteristics of the liposomes can be altered
depending on the drug and site of action.

14. Disadvantages of Liposomes
 Physical/ chemical stability
 Very high production cost
 Drug leakage/ entrapment/ drug fusion
 Sterilization
 Short biological activity or Biological Half life
 Oxidation of bilayer phospholipids and low solubility
 Rate of release and altered bio distribution
 Low therapeutic index and dose effectiveness
 Overcoming resistance
 Extensive clinical and laboratory research to a certain long circulating liposome
 Repeated iv administration problems

15. FORMULATION OF LIPOSOMES
Liposomes are mainly prepared by,
 Active loading ( Remote loading )
 Passive loading
1.Mechanical dispersion method
2.Solvent dispersion method
3.Detergent removal method

16. 1.Mechanical dispersion method
Lipid film hydration by hand shaking non-hand shaking and freeze drying
Microfluidizer techniques for Micro emulsification
Sonication
French pressure cell
Membrane extrusion
Dried reconstituted vesicles
Fusion method
Freeze thawed Technique
2.Solvent dispersion method
Ethanol injection
Ether injection
De emulsion vesicles
Double Emulsion Method
Rapid solvent exchange method
Reverse phase evaporation vesicles
3.Detergent removal method
Detergent removal from mixed micelles by
Dialysis
Column chromatography
Dilution
Reconstituted sendai virus enveloped vesicles

17. 1.Mechanical dispersion method
a. LIPID FILM HYDRATION BY HAND-SHAKING, NON-HAND SHAKING
Step-1: Preparation of film for hydration:
1) Lipid + organic solvent (chloroform/ chloroform: methanol) 10-20mg lipid/ml Mix
thoroughly
2) Solvent removal– Small volume (< 1ml)= dry N2/Argon stream, Large volume=
Rotary evaporation thin lipid film.
3) Lipid film is dried to eliminate residual organic solvent by placing in vacuum over
night.
4) Lipid film can also be prepared by freezing in dry ice/ dry iceacetone/ alcohol bath.
5) The frozen lipid cake is placed in vacuum pump and lyophilized until dry (1-3
days).
6) Dry lipid films removed from vacuum, container closed tightly, taped and stored
frozen.

18. Step-2: Hydration of lipid film:
1) The dried lipid film can be hydrated by addition of aqueous medium, followed by
agitation (1 hr) and over night stand.
2) Temperature of hydrating medium should be above “Tc”.
3) Hydration media– buffer solution, saline, 5% dextrose, 10% sucrose.
4) The obtained product- LMV suspension is downsized.

19. b. MICROFLUIDIZER TECHNIQUES FOR MICRO EMULSIFICATION
1. “Micro Fluidizer” is used to prepare small ULV/ MLVs from concentrated lipid
dispersion.
2. The lipids are introduced into fluidizers, either as a dispersion of large MLVs
or as a slurry of unhydrated lipids in organic medium.
3. Microfluidizer pumps the fluid at very high pressure (10,000psi, 600-700 bar)
through a 5um orifice.
4. Then it is forced along defined micro channels, which direct two streams of
fluid to collide together at right angles at a very high velocity, thereby
affecting an efficient transfer of energy.
5. The fluid collected of be recycled through the pump and interaction chamber
untill vesicles of the spherical dimension are obtained.
Adv: Samples with high % of lipids can be easily treated.

20. c. SONICATION:
2 Types
1.Bath sonicator
2.Probe sonicator
1.Bath sonicator
 Large volume of diluted lipids are processed.
 Less or no contamination.
 At high energy levels, average size of vesicles is further reduced.
 Exposure of MLV’s to ultrasonic irradiations is the most widely used method for
producing small vesicles.
 As chances of contamination are likely to occur in bath sonicator is widely used.

21. 2.Probe sonicator
 Small volume of diluted lipids are processed.
 Chances of contamination.
 At high energy levels, average size of vesicles is further reduced.
 Exposure of MLV’s to ultrasonic irradiations is the most widely used method for
producing small vesicles.
 As chances of contamination are likely to occur in probe sonicator is widely
used.

22. d. FRENCH PRESSURE CELLS (ULV/OLV)
 Method developed by Barenholtz & Hamilton.
 Very useful method in which extrusion of preformed large liposomes in a
French Pressure under very high pressure is carried out.
 This technique yields ULV’s/OLV’s of intermediate
size(3080nm/depending upon applied pressure).
 Liposomes are more stable.
 Free from structural defects.
 Leakage problem is also less.
 However it has high production cost

23. e. MEMBRANE EXTRUSION:
1. Used for preparation of LUVs and MLVs.
2. The size of liposomes is reduced by gently passing them through polycarbonate
membrane filter of defined pore size at lower pressure
3. Before extrusion LMV are disrupted by freeze-thaw cycles/ pre-filtering through
large pore size (0.2-1 µm).
4. Filter with 100nm pores yield LUV of 120-140nm.

24. f. DRIED-RECONSTITUTED VESICLES:
• This method involves freeze drying the dispersion of empty SUV followed
by rehydration with aqueous fluid, which have material to be entrapped.
• Useful for preparation of small uni-lamellar and oligo lamellar vesicles.
Adv: High entrapment of water soluble components and bioactives.
g. FUSION METHOD:
 This method protects lipids and entrapped material from harmful
physicochemical environment.
 Used for preparation of ULV in large quantities.
 Fusogenic agents are used for fusion of SUV for increasing entrapment
efficiency.
 Alteration in pH increases surface charge density of lipid bilayer and
brings on spontaneous vesiculation.

25. h. FREEZE THAW SONICATION (FTS)
Freeze SUV dispersion
thaw at room temperature for 15 minutes
sonicate
rupture of SUV’s occur
Formation of liposomes

26. 2. SOLVENT DISPERSION METHODS
A. ETHANOL INJECTION METHOD
Lipids + ethanol
Rapidly inject through a fine needle
Saline buffer containing materials to be entrapped
dissolution of ethanol
Formation of SUV’s.

27. B. ETHER INJECTION METHOD
Lipid + ether
slowly injecting through a narrow needle
vapourize temperature at 60˚C
production of SUV’s
 Less risk of oxidation as ether is free of peroxides.
 Low efficiency.
 Long time needed for production.

28. C. DE EMULSIFICATION METHOD
Generally liposome is made up in 2 steps
 First the inner leaflet of the bilayar.
 Then the outer half.
 Aqueous medium + material to be entrapped
 High volume organic solution of lipid
 Agitated mechanically
 Break aqueous Medium to water droplets
 W/O emulsion
 Stabilized by phospholipid monolayer.

29. D. DOUBLE EMULSION METHOD:
1. The organic solution, which already contains water droplets (W/O) is introduced in
to excess aqueous medium followed by mechanical dispersion causing phase
inversion (O/W).
2. The (W/O/W) multi component vesicle is formed by double emulsion.
3. Two aqueous compartments being separated from each other by two phospholipid
monolayer.
4. The hydrophobic surfaces of monolayers (tails) face each other, with a thin film of
organic solvent.
5. Removal of organic solvent results in formation of intermediated sized unilamellar
vesicles.

30. E. RAPID SOLVENT EXCHANGE METHOD:
• Lipid solution in organic solvent is passed through an orifice of syringe by
means of vacuum in to a tube containing aqueous buffer placed on vortex.
• The organic solvent vaporizes due to vacuum before contacting aqueous
phase.
• The lipid mixture precipitates very quickly in aqueous buffer forming
liposomes.
F. REVERSE PHASE EVAPORATION:
1. Mixture of 2 phases (Aq+Org) subjected to bath sonication.
2. Droplets formed are dried to semisolid gel in rotary evaporator under
reduced pressure.
3. In this stage mono layer of phospholipids surround water compartment.
4. Mechanical shaking with vortex shaker collapses few water droplets &
lipid mono layers. 5. Collapsed lipid monolayers form outer layer for stable
vesicles thus forming bilayer SUV.

31. Reverse phase evaporation vesicles
Lipid + organic solvent + aqueous solution
mix well
sonicate
formation of w/o emulsion
evaporate to remove the organic solvent
Lipids form a phospholipid bilayer
vigorous shaking
water droplets collapse
formation of LUV’s.

33. Evaluation of liposomes
The liposomes prepared by various techniques are to be evaluated for their physical
properties, has these influence the behavior of liposomes in vivo.
Physical properties
1. Particle size
Both particle size and particle size distribution of liposomes influence their
physical stability. These can be determined by the following method.
a) Laser light scattering
b) Transmission electron microscopy

34. 2. Surface charge
o The positive, negative or neutral charge on the surface of the liposomes is
due to the composition of the head groups.
o The surface charge of liposomes governs the kinetic and extent of
distribution in vivo, as well as interaction with the target cells.
o The method involved in the measurement of surface charge is based on
free-flow electrophoresis of MLVs.
o It utilizes a cellulose acetate plate dipped in sodium borate buffer of pH 8.8.
o About 5N moles of lipid samples are applied on to the plate, which is then
subjected to electrophoresis at 4 0c for 30 mins.
o The liposomes get bifurcated depending on their surface charge.
o This technique can be used for determining the heterogeneity of charges in
the liposome suspension as well as to detect any impurities such as fatty
acids.

35. 3. Percent drug encapsulated
• Quantity of drug entrapped in the liposomes helps to estimate the behavior of the
drug in biological system.
• Liposomes are mixture of encapsulated and unencapsulated drug fractions.
• The % of drug encapsulation is done by first separating the free drug fraction from
encapsulated drug fraction.
• The encapsulated fraction is then made to leak off the liposome into aqueous
solution using suitable detergents.
• The methods used to separate the free drug from the sample are:
Mini column centrifugation method
Protamine aggregated method

36. 4. Phase behavior
• At transition temperature liposomes undergo reversible phase transition
• The transition temperature is the indication of stability permeability and also
indicates the region of drug entrapment
• Done by DSC
5. Drug Release Rate
 The rate of drug release from the liposomes can be determined by in vivo
assays which helps to predict the pharmacokinetics and bioavailability of the
drug. However in vivo studies are found to be more complete.
 Liposome encapsulating the tracer insulin are employed for the study. This
insulin is preferred, as it is released only in the ECF and undergoes rapid renal
excretion of the face tracer coupled to the degradation rate constant o the tracer
released from the liposomes.

37. APPLICATIONS
1. The therapeutic value of liposomes as drug carriers, particularly for
anticancer, antifungal, and antibacterial agents.
2. As anticancer , cytotoxic drugs like Cytarabine, alkylating agents.
3. As vaccine adjuvants i.e. when administered by IM route, they slowly
release the antigens and accumulate in lymph nodes.
4. In ophthalmic drug delivery systems, Idoxuridine used in acute & chronic
keratitis.
5. Sustained release system of systemically or locally administered
liposomes.
Eg: biological proteins or peptides such as vasopressin.
6. Site specific targeting: in certain cases liposomes with surface attached
ligands can bind to target cells (‘key and lock’ mechanism).
Eg: anti infectors & anti inflammatory drugs.