SUBMITTED BY: RUHIT ASHRAF
SUBMITTED TO: AMANDEEP
Deptt. of PHARMACY,
School of PHARMACEUTICAL
AND HEALTH CARE SCIENCES.
MOLECULAR PHARMACEUTICS – MPH 201 T
Targeting Methods: introduction preparation and
evaluation. Nano Particles &
Liposomes:Types,preparation and evaluation.
. A special form of drug delivery system
where the pharmacologically active agent or
medicament is selectively targeted or
delivered only to its site of action or
absorption and not to the non-target organs
or tissues or cells.
. Targeted drug delivery implies for selective and
effective localization of pharmacologically active
moiety at pre identified (preselected) target in
therapeutic concentration, while restricting its
access to non-target normal cellular linings, thus
minimizing toxic effects and maximizing
• Drug administration protocols may be
• Drug quantity may be greatly reduced as
well as the cost of therapy.
• Drug concentration in the required sites
can be sharply increased without negative
effects on non-target compartments.
• Rapid clearance of targeted systems.
• Immune reactions against intravenous
administered carrier systems.
• Insufficient localization of targeted systems
into tumour cells.
• Diffusion and redistribution of released
. Targeted drug delivery system should be-
biochemically inert (non-toxic), non-
. Both physically and chemically stable in vivo
and in vitro.
. Restrict drug distribution to target cells or
tissues or organs and should have uniform
. Controllable and predictable rate of drug
. Nanoparticles (NPs) are defined as
particulate dispersions or solid
particles drug carrier that may or
may not be biodegradable.
. The drug is dissolved, entrapped,
encapsulated or attached to a
. The term nanoparticle is a
combined name for both
nanospheres and nanocapsules.
. Drug is confined to a cavity surrounded
by a unique polymer membrane called
nanocapsules, while nanospheres are
matrix systems in which the drug is
physically and uniformly dispersed.
Advantages of Nanoparticles
.Nanoparticles have many significant
advantage over conventional and traditional
drug delivery system.
.Nanoparticles are control and sustain release
form at the site of localization, they alter
organ distribution of drug compound.
.They enhance drug circulation in blood,
bioavailability, therapeutic efficacy and
reduce side effects.
. Nanoparticles can be administer by various
routes including oral, nasal, parenteral,
. In the tiny areas of body nanoparticles shows
better drug delivery as compare to other
dosage form and target to a particular cell
type or receptor.
. Nanoparticle enhance the aqueous solubility
of poorly soluble drug, which improves
bioavailability of drug.
. As a targeted drug carrier nanoparticles
reduce drug toxicity and enhance efficient
. Useful to diagnose various diseases.
. Enhanced stability of ingredients.
1. Solvent Evaporation.
. Solvent evaporation method first developed
for preparation of nanoparticles.
. In this method firstly nanoemulsion
. Polymer dissolved in organic solvent
(dichloromethane, chloroform or ethyl
. Drug is dispersed in this solution.
. Then this mixuture emulsified in an aqueous
phase containing surfactant (polysorbates,
. This make an oil in water emulsion by using
mechanical stirring, sonication, or micro
fluidization (high-pressure homogenization
through narrow channels).
. Representation of the solvent – evaporation
Double Emulsification Method
. Emulsification and evaporation method
have limitation of poor entrapment of
hydrophilic drugs, hence double
emulsification technique is used.
. Firstly w/o emulsion prepared by addition of
aqueous drug solution to organic polymer
solution with continuous stirring.
. This prepared emulsion another aqueous
phase with vigorous stirring, resultant
w/o/w emulsion prepared.then organic
solvent removed by high centrifugation.
Emulsions - Diffusion Method.
• This method patent by Leroux et al it is
modified form of salting out method.
• Polymer dissolved in water-miscible solvent
(propylene carbonate, benzyl alcohol), this
solution saturated with water.
• Polymer-water saturated solvent phase is
emulsified in an aqueous solution containing
stabilizer. Then solvent removed by
evaporation or filtration
. Representation of the emulsification diffusion
Nano precipitation method.
. In this method precipitation of polymer and
drug obtained from organic solvent and the
organic solvent diffused in to the aqueous
medium with or without presence of
. Firstly drug was dissolved in water, and then
cosolvent (acetone used for make inner phase
more homogeneous) was added into this
• Then another solution of polymer (ethyl
cellulose, eudragit) and propylene glycol with
chloroform prepared, and this solution was
dispersed to the drug solution.
• This dispersion was slowly added to 10 ml of
70% aqueous ethanol solution.
• After 5 minutes of mixing, the organic solvents
were removed by evaporation at 35°under
normal pressure, nanoparticles were
separated by using cooling centrifuge (10000
rpm for 20 min), supernatant were removed
and nanoparticles washed with water and
dried at room temperature in a desicator.
. Drug and protein solution (2% w/v) incubated
for one hour at room temperature and pH
adjusted to 5.5 by using 1M HCl.
. In this solution ethanol was added in 2:1 ratio
(v/v) in a controle rate 1ml/min.
. Resultant coacervate hardened with 25%
glutaraldehyde (1.56 μg/mg of protein) for 2
hours which allow cross-linking of protein.
• Rotary Vacuum evaporation at reduced
pressure organic solvents were removed then
nanoparticle were collected and purified by
centrifugation at four degree centigrade.
• Pellets of nanoparticles were then suspended
in phosphate buffer (pH 7.4; 0.1 M) and
lyophilized with mannitol.
• Salting Out Method.
• Salting out method is very close to solvent-
• This technique is based on the separation of
water-miscible solvent from aqueous solution
by salting out effect.
• In this method toxic solvents are not used.
• Generally acetone is used because it is totally
miscible with water and easily removed.
. Polymer and drug dissolved in a solvent which emulsified
into a aqueous solution containing salting out agent
(electrolytes, such as magnesium chloride and calcium
chloride, or nonelectrolytes such as sucrose).
Dialysis is an effective method
for preparation of
In this method firstly polymer
(such as Poly(benzyl-
oxide), Poly(lactide) and drug
dissolved in a organic solvent.
This solution added to a
dialysis tube and dialysis
Evaluation Parameters Of
Yield of Nanoparticles.
% Yield = amount of nanoparticle 100
amount of drug + polymer
. The yield of nanoparticles was determined by
comparing the whole weight of nanoparticles
formed against the combined weight of the
copolymer and drug.
Particle Size and Zeta Potential.
• Value of Particle size and Zeta Potential
prepared nanoparticles determined by using
• Surface morphology study carried out by
Scanning Electron Microscopy (SEM) of
• Polydispersity index of prepared nanoparticles
was carried out by using Malvern Zetasizer.
In-vitro release Study.
• In-vitro drug release studies were performed in
USP Type II dissolution apparatus at rotation
speed of 50 rpm.
• The prepared immersed in 900ml of phosphate
buffer solution in a vessel, and temperature was
maintained at 37±0.20°C.
• Required quantity 5ml of the medium was
withdrawn at specific time periods and the same
volume of dissolution medium was replaced in the
flask to maintain a constant volume.
• The withdrawn samples were analyzed using UV
. Study of nanoparticles were fitted with various
kinetic equation like
. zero order (cumulative % release vs. time)
. first order (log % drug remaining vs time)
. Higuchi s model (cumulative % drug release vs.‟
square root of time).
Stability of Nanoparticles.
• Stability studies of prepared nanoparticles
determined by storing optimized formulation at
4°C ±1°C and 30°C ± 2°C in stability chamber for
• The samples were analyzed after a time period
like at 0, 1, 2, and 3 months for their drug
content, drug release rate (t-50%) as well as any
changes in their physical appearance.
TYPES OF NANOPARTICLES
. Nanoparticles can be classified into different
types according to the size, morphology,
physical and chemical properties. Some of
• Polymeric nanoparticles.
• Lipid-based nanoparticles.
1. Carbon-based nanoparticles:
. Carbon-based nanoparticles include two main
a. Carbon nanotubes (CNTs).
• CNTs are nothing but graphene sheets rolled
into a tube. These materials are mainly used for
the structural reinforcement as they are 100
• Fullerenes are the allotropes of carbon having a
structure of hollow cage of sixty or more carbon
atoms. The structure of C-60 is also called
• Ceramic nanoparticles are inorganic solids
made up of oxides, carbides, carbonates and
• These nanoparticles have high heat resistance
and chemical inertness.
• They have applications in photocatalysis,
photodegradation of dyes, drug delivery, and
• Metal nanoparticles are prepared from metal
• These nanoparticles can be synthesized by
chemical, electrochemical, or photochemical
• These nanoparticles have applications in
detection and imaging of biomolecules and in
environmental and bioanalytical applications.
• Semiconductor nanoparticles have properties
like those of metals and non-metals.
• These particles have wide bandgaps, which
on tuning shows different properties.
• They are used in photocatalysis, electronics
devices, photo-optics and water splitting
• Some examples are Gan,Gap,Inp etc.from
• Polymeric nanoparticles are organic based
• Depending upon the method of preparation,
these have structures shaped like
nanocapsular or nanospheres.
• A nanosphere particle has a matrix-like
structure whereas the nanocapsular particle
has core-shell morphology.
• Lipid nanoparticles are generally spherical in
shape with a diameter ranging from 10 to
• It consists of a solid core made of lipid and a
matrix containing soluble lipophilic molecules.
• The external core of these nanoparticles is
stabilized by surfactants and emulsifiers.
. A Lamella is a flat plate like structure that
appears during the formation of liposomes.
. The phospholipids bilayer first exists as a
lamella before getting converted into spheres.
. Several lamella of phospholipids bilayers are
stacked one on top of the other during
formation of liposomes to form a
. Small unilamellar vesicle.
. Large unilamellar vesicle.
. Multi lamellar vesicle.
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.
. Improved pharmacokinetic effects.
. Site avoidance effect (avoids non-target tissues).
. Physical/ chemical stability.
. Very high production cost.
. Drug leakage/ entrapment/ drug fusion.
. Short biological activity / t ½.
.Oxidation of bilayer phospholipids.
. Overcoming resistance.
.Repeated iv administration problems.
Preparation of liposomes
Methods of liposome preparation
. Passive loading: Involves loading of the entrapped
agents before or during the manufacturing procedure.
. Active or remote loading: Certain types of compounds
with ionisable groups and those with both
manufacturing procedure lipid and water solubility can
be introduced into the liposomes after the formation of
the intact vesicles.
Methods of liposome preparation
Passive Loading techniques.
. It comprises of three different types.
1.Mechanical dispersion methods.
2. Solvent dispersion method.
3.Detergent removal method.
1. Mechanical dispersion method
. Lipid film hydration by hand shaking non- hand
shaking and freeze drying.
.French pressure cell.
.Dried reconstituted vesicles.
.Freeze thawed liposomes
Evaluation of liposomes
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.
2. Surface charge
• The positive, negative or neutral charge on
the surface of the liposomes is due to the
composition of the head groups.
. The surface charge of liposomes governs the
kinetic and extent of distribution in vivo, as
well as interaction with the target cells.
. The method involved in the measurement of
surface charge is based on free-flow
electrophoresis of MLVs.
• 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.
3. Percent drug encapsulated
• Quantity of drug entrapped in the liposomes
helps to estimate the behavior of the drug in
• 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.
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
• Liposomes as drug or protein delivery vehicles.
• Liposome in antimicrobial, antifungal(lung
therapeutics) and antiviral (anti HIV) therapy.
• In tumor therapy.
• In gene therapy.
• In immunology.
• Liposomes as artificial blood surrogates. •
Lecture No. 1/References
•Sovan Lal Pal, Utpal Jana, P. K. Manna, G. P. Mohanta, R.
Manavalan, Nanoparticle: An overview of preparation and
characterization, Journal of Applied Pharmaceutical Science 2011;
•Gayatri Khosla, Lakshmi Goswami, Preeti Kothiyal, Sayantan
Mukhopadhyay, Nanoparticles: A Novelistic Approach for CNS
disorders, Journal of Advanced Pharmaceutical Sciences 2012; 2:2:
•Abhilash M., Potential applications of Nanoparticles, International
Journal of Pharma and Bio Sciences 2010; 1:1: 1- 12.