INTRODUCTION PREPARATION
& EVALUATION OF
NANOPARTICLES

I M PHARM

DEPT. OF PHARMACEUTICS

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WHAT ARE NANOPARTICLES?

❑Nano derives from the Greek word “Nanos” which means Dwarf or
Extremely small. It can be used as a prefix for any unit to mean a billionth
of that unit.

❑A nanometer is a billionth of a meter.

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DEFINITION:

➢ Nanoparticles are solid colloidal particles ranging from 1to100nm in size.

➢ They are composed of synthetic or semi synthetic polymers carrying drugs
or proteinaceous substances, i.e. antigen(s).

➢ Drugs are entrapped, or encapsulated in the polymer matrix particulates or
solid solutions or may be bound to particle surface by physical adsorption
or in chemical form.

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CONCEPT:

The basic Concept involved is :

✓ Selective and Effective Localization of pharmacologically active
moiety at preselected target(s) in therapeutic concentration,,

✓ Provided restriction of it’s access to non-target normal tissues and
cells.

✓ Nanoparticles are mainly taken by : Reticuloendothelial System (RES),
after the administration.

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✓ Hence are useful to carry drugs to the liver and to cells that are
phagocytically active.

✓ By modifying the surface characteristics of the nanoparticles it is possible
to enhance the delivery of drugs to spleen relative to the liver.

✓ Distribution of the nanoparticles in the body may be achieved possibly by :
Coating of nanoparticles with certain Serum components, Attachment of
antibodies or sulfoxide groups and the use of Magnetic nanoparticles.

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The term nanoparticle is a combined name for both nanospheres and
nanocapsules.

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Nanospheres and Nanocapsules

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DIFFERENT TYPES OF NANOPARTICLES
Solid Lipid Nanoparticles
Polymeric Nanoparticles
Ceramic Nanoparticles
Hydrogel Nanoparticles
Copolymerized Peptide Nanoparticles
Nanocrystals and Nanosuspensions
Nanotubes And Nanowires
Functionalized Nanocarriers
Nanospheres
Nanocapsules

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❖Solid lipid Nanoparticles:
 New type of colloidal drug carrier system for i.v.
 Consists of spherical solid lipid particles in the nm range,

dispersed in water or in aqueous surfactant solution.

❖Polymeric nanoparticles (PNPs) :
 They are defined as particulate dispersions or solid particles with
size in the range of 10-1000nm.Composed of synthetic or semi-
synthetic Polymers.
 Biodegradable polymeric nanoparticles Polylactic acid (PLA),
polyglycolic acid (PGA), Polylactic – glycolic acid (PLGA), and
Polymethyl methacrylate (PMMA) Phospholipids Hydrophobic core

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❖Ceramic Nanoparticles:
These are the nanoparticles made up of inorganic (ceramic)
compounds silica, ( Inorganic/metal) titania and alumina.
Exist in size less than 50 nm, which helps them in evading deeper
parts of the body.

❖Hydrogel nanoparticles:
Polymeric system involving the self-assembly and self aggregation
of natural polymer amphiphilic cholesteryl pullulan , cholesteryl
dextran and agarose cholesterol groups provide cross linking
points.

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❖Copolymerized Peptide Nanoparticles:
Drug moiety is covalently bound to the carrier instead of being
physically entrapped.

❖Nanocrystals And Nanosuspensions:
Pure drug coated with surfactant, Aggregation of these particles in
crystalline form .Drug powder dispersed in aqueous surfactant
solution.

❖Functionalized Nanocarriers:
Biological materials like proteins, enzymes, peptides etc… are
being utilized as a carriers for the drug delivery.

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Advantages of nanoparticles
• Nanoparticles can be administer by various routes including oral, nasal,
parenteral, intra-ocular etc.
• Due to small particle size nanoparticles overcome resistance by
physiological barriers in the body and easily penetrates to cell walls, blood
vessels, stomach epithelium and blood–brain barrier.
• As a targeted drug carrier nanoparticles reduce drug toxicity and
enhance efficient drug distribution
• Polymeric nanoparticle an ideal drug delivery system for cancer therapy,
vaccines, contraceptives and antibiotics.
• Useful to diagnose various diseases
• Enhanced stability of ingredients
• Prolonged shelf life
• Used in dental surgery also as filling the tiny holes in teeth. 14
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Disadvantages Of Nanoparticles
• Small size & large surface area can lead to particle aggregation.
• Physical handling of nano particles is difficult in liquid and dry forms.
• Limited drug loading.
• Toxic metabolites may form.
• Difficult to maintain stability of dosage form.
E.g.: Resealed erythrocytes stored at 40C.

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IDEAL CHARACTERISTICS
» It should be biochemical inert , non toxic and non-immunogenic.
» It should be stable both physically and chemically in in-vivo & in-vitro
conditions.
» Restrict drug distribution to non-target cells or tissues or organs &
should have uniform distribution.
» Controllable & Predicate rate of drug release.
» Drug release should not effect drug action
» Specific Therapeutic amount of drug release must be possessed
» Carriers used must be biodegradable or readily eliminated from the
body without any problem and no carrier induced modulation in disease
state.
» The preparation of the delivery system should be easy or reasonable

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w»ww .SDuilomMix.pcomle, reproducible & cost effective.

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METHODS OF PREPARATION
A : Amphiphilic Macromolecules Cross Linking Methods
1) Crosslinking in W/O Emulsion
2) Emulsion chemical dehydration
3) Phase Separation in Aqueous Medium (Desolvation)
4) pH induced Aggregation
B : Polymerization Methods
1) Emulsion polymerization
2) Dispersion polymerization
3) Interfacial condensation polymerization
4) Interfacial complexation
C : Polymer precipitation methods
1) Solvent evaporation/extraction
2) Solvent displacement (nanoprecipitation) 19

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3) Salting out

A. AMPHIPHILIC MACROMOLECULES CROSS
LINKING METHODS

• Nanoparticles can be prepared from Amphiphilic macromolecules, proteins and
polysaccharides (which have affinity for aqueous and lipid solvents).

• The method involves Aggregation of Amphiphiles followed by 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,
extraction, or diffusion of solvent, amphiphiles are aggregated as tiny particulates &
subsequently rigidized via chemical cross-linking.

1) Cross linking in W/O Emulsion
▪ The cross linking method is exhaustively used for the nano-encapsulation of drugs20

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▪ The method involves the emulsification of bovine serum albumin (BSA) or human
serum albumin (HSA) or protein aqueous solution in oil using high-pressure
homogenization or high frequency sonication.

▪ The W/O emulsion so formed is then poured into preheated oil (temp. above 1000C),
which is then stirred for a specified time in order to denature & aggregate the protein
contents of aqueous pool completely & to evaporate the water.

▪ Proteinaceous sub-nanoscopic particles are thus formed where the size of internal
phase globules mainly determines the ultimate size of particulates.

▪ The particles are finally washed with an organic solvent to remove any adherent or
adsorbed oil traces & subsequently collected by centrifugation.

▪ The crucial factors which governs the size & shape of nanoparticles are mainly
emulsification energy & temperature (used for denaturation & aggregation).

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2) Emulsion Chemical Dehydration

» Stabilization can also be achieved by emulsion chemical dehydration.
» Hydroxy propyl cellulose solution in chloroform is used as a continuous
phase, &
» A chemical dehydrating agent, 2,2, di-methyl propane is used to
disperse into the internal aqueous phase to form an Emulsion.
» ADV: The method avoid coalescence of droplets and could produce
nanoparticles of smaller size (300nm).

3) Phase Separation in Aqueous Medium (Desolvation)
» The protein or polysaccharide from an aqueous phase can be
Desolvated by :
˃ A) pH change
˃ B) Change in temperature 23
˃ww wC.Dul)oM Aix.codmdition of appropriate counter ions e.g. alginate

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4) pH Induced Aggregation
Gelatin Nanospheres were prepared by :

➢ Gelatin & tween 20 were dissolved in aq. phase & pH of the solution was
adjusted to optimum value.

➢ The clear solution so obtained was heated to 400C followed by its
quenching at 40C for 24hrs & subsequently left at ambient temperature
for 48hrs.

➢ The sequential temperature treatment resulted into a colloidal dispersion
of aggregated gelatin. The aggregates were finally cross linked using
glutaraldehyde as cross linking agent. The nanospheres thus resulted were
of 200 nm average size with uniform dispersity.

➢ The optimum pH range for ideal & uniform preparation of gelatin
nanospheres was 5.5- 6.5.

➢ pH value below 5.5 produced no aggregation while above 6.5 an 25
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uncontrolled aggregation led to the formation of larger nanospheres

B. POLYMERIZATION BASED METHODS
The polymers used for nanosphere preparation include;

Poly(methylmethacrylate)
Poly(butyl cyano-acrylate)
Poly(acrylamide)
N-N’methylene-bis-acrylamide

1) Emulsion Polymerization
• Emulsion Polymerization is a method in which the monomer to be polymerized is

emulsified in a non-solvent phase.
• The process can be conventional or inverse, depending upon the nature of the continuous

phase in the emulsion.
In Conventional, the continuous phase is aqueous (O/W)
In Inverse, the continuous phase is organic (W/O)
• Two different mechanisms were proposed for the emulsion polymerization process

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a) Micellar nucleation & polymerization

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b) Homogenous nucleation & polymerization

a) Micellar nucleation and polymerization
✓ In this the monomer is emulsified in non-solvent phase using surfactant molecules.
✓ This leads to the formation of Monomer- swollen micelle & Stabilized monomer

droplets.
✓ Monomer-swollen micelle have sizes in nanometric range and have much larger

surface area compared to monomer droplet
✓ Polymerization reaction proceeds through nucleation and propagation stage in

presence of chemical or physical initiator.

✓ Energy provided by initiator creates free monomers in continuous phase, which then
collide with surrounding unreactive monomers and initiate polymerization chain
reaction.

✓ The monomer molecule reaches the micelle by diffusion from the monomer droplets
through continuous phase, thus allowing polymerization to progress within micelles.
Here monomer droplets act as reservoirs of monomers.

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As monomer is slightly soluble in surrounding phase, it diffuses from monomer droplets and reach monomer
micelles through continuous phase. Thus polymerization takes places in MICELLES.

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b) Homogenous nucleation and polymerization
✓ In this method monomer is sufficiently soluble in continuous outer phase. Nucleation

and polymerization can directly occur in this phase leading to formation of primary
chains called oligomers.

✓ In this both micelle and droplets act as monomers reservoir throughout polymer chain
length.

✓When oligomers reach certain length, they precipitate and form primary particles and
stabilized by surfactant molecules provided by micelle and droplets in which the drug
will entrapped to form nanoparticles.

✓ The polymerization rate is dependent on the pH of the medium.
✓ Anionic polymerization takes place in micelle after diffusion of monomer molecules
through the water phase and is initiated by negative charged compound
✓ At neutral pH the rate of polymerization is extremely fast.

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2) Dispersion Polymerization

➢ In emulsion polymerization monomer is emulsified in an immiscible phase using
surfactant. In case of dispersion polymerization monomer is dissolved in an aqueous

medium which acts as precipitant for polymer
➢ The monomer is introduced into the dispersion medium.
➢ Polymerization is initiated by adding a catalyst & proceeds with nucleation phase

followed by growth phase.
➢ Nucleation is directly induced in aqueous monomer solution and presence of

stabilizer or surfactant is not necessary for the formation of stable nanospheres.

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3) Interfacial polymerization
▪ The preformed polymer phase is finally transformed to an embryonic

sheath.
▪ The polymer & drug are dissolved in a volatile solvent. The solution is

poured into a non solvent for both polymer & core phase.
▪ The polymer phase is separated as a coacervate phase at o/w interface.
▪ The mixture turns milky due to formation of nanocapsules.
▪ This method is used for proteins, enzymes, antibodies, & cells
▪ The size of nanocapsules ranges from 30-300nm

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4) Interfacial Complexation

 The method is based on the process of microencapsulation
introduced by Lin & Sun 1969.
 In case of nanoparticles preparation, aqueous polyelectrolyte
solution is carefully dissolved in reverse micelles in an apolar bulk
phase with the help of an appropriate surfactant.
 Subsequently, competing polyelectrolyte is added to the bulk,
which allows a layer of insoluble polyelectrolyte complex to
coacervate at the interface.

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C. POLYMER PRECIPITATION METHODS

The polymer precipitation is done by
✓ Increasing the solubility of organic solvent in the external medium by
adding an alcohol (isopropanol)
✓ By incorporating water into ultra emulsion(to extract solvent)
✓ By evaporation of solvent at room temp. by using vacuum
✓ Using an organic solvent which is completely soluble in the continuous
aq. phase(acetone)nanoprecipitation

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1) SOLVENT EVAPORATION METHOD

Nanoparticles preparation using Emulsion solvent evaporation
method

➢ This method involves the formation of a conventional O/W emulsion
between a partially water miscible solvent containing the stabilizer.

Ex: PLGA nanospheres
➢ The polymer is solubilized in a solvent (chloroform) and dispersed in

gelatin solution by sonication to yield O/W emulsion. The solvent is
eliminated by evaporation. For evaporation homogenizer is used which
breaks the initial coarse emulsion in nanodroplets yielding nanospheres.

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2) SOLVENT DISPLACEMENT METHOD (Nanoprecipitation)

o It is based on interfacial deposition of a polymer following displacement of
a semi polar solvent miscible with water from a lipophilic solution

o The organic solvent diffuses instantaneously to the external aq. Phase
inducing immediate polymer precipitation because of complete miscibility
of both the phases

o If drug is highly hydrophilic it diffuses out into the external aq. phase while
if drug is hydrophobic it precipitates in aq. medium as nanocrystals

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3) SALTING OUT TECHNIQUE
• Salting out is based on the separation of a water-miscible solvent from
aqueous solution via a salting-out effect.
• Polymer and drug are initially dissolved in a solvent which is subsequently
emulsified into an aqueous gel containing the salting out agent (electrolytes,
such as magnesium chloride and calcium chloride, or non- electrolytes such
as sucrose) and a colloidal stabilizer such as polyvinylpyrrolidone (PVP) or
hydroxyethylcellulose.
• This O/W emulsion is diluted with a sufficient volume of water or
aqueous solution to enhance the diffusion of solvent into the aqueous phase,
thus inducing the formation of nanospheres.
• It is different from nanoprecipitation method as in nanoprecipitation
polymeric solution is completely miscible with the external phase. But in this
method the miscibility of both the phase is prevented by the saturatio4n2 of

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external aqueous phase with PVA and Magnesium chloride.

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NOVEL NANOPARTICULATE SYSTEMS

Solid Lipid Nanoparticles:
These are sub-micron colloidal carriers (50-100nm) which are composed of
physiological lipid dispersed in water or in a aqueous surfactant solution.

Micro emulsion technique was used for the production of solid lipid
nanoparticles

Homogenization method at higher pressure for either melted or solid
lipids has been suggested to obtain SLN.

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Advantages of SLN

✓ Small size and relatively narrow size distribution which provide
biological opportunities for site specific drug delivery by SLNs.

✓ 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.

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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.

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Hot Homogenization Technique
Melting of the lipid

Dissolution of drug in melted lipid

Mixing of preheated dispersion medium & drug lipid melt

Premix using stirrer to form coarse pre-emulsion

High pressure homogenization at a temp. above the lipids melting point

o/w nanoemulsion

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wwSw.DoulolMiidx.ciomfication of the nanoemulsion by cooling down to room temp. to form

SLN

Cold Homogenization Technique

Melting of the lipid

Dissolution /solubilization of drug in melted lipid

Solidification of drug loaded lipid in liquid nitrogen or dry ice

Grinding in a powder mill(50-100μm particles)

Dispersion of lipid in the cold aqueous dispersion medium

Solid lipid nanoparticles 48
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NANOCRYSTALS & NANOSUSPENSIONS
Nanocrystals and Nanosuspensions are two recently introduced aspects

to drug delivery research.
The basic theme is to convert micronized drug powders to drug

nanoparticles.

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PHARMACEUTICAL ASPECTS OF NANOPARTICLES
Should be free from potential toxic impurities
Should be easy to store and administer
Should be sterile if parenteral use is advocated.

Three important process parameters are performed before releasing them for
clinical trials:
o Purification
o Freeze drying
o Sterilization.

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Cross-flow filtration technique
This method has been suggested for the

purification of nanoparticles and the
method can be scaled up from an
industrial stand point.

In this method the suspension is filtered
through membranes with direction of fluid
being tangential to surface of membrane.

Depending on the type of membrane
used either microfiltration or ultra
filtration can be performed.

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Freeze drying of nanoparticles
It includes freezing of nanoparticle suspension & sublimation of water to
produce free flowing powder.

Advantages are
• Prevention from degradation & or solubilization of the polymer
• Prevention from drug leakage, drug desorption, drug degradation
• Nanocapsules containing oily core may be processed in the presence of
mono or disaccharides (glucose or sucrose)
• Readily dispersible in water without modifications in their
physicochemical properties

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Sterilization of Nanoparticles
➢ Nanoparticles intended for parenteral use should be sterilized to be

pyrogen free before animal or human use.
➢ Sterilization in nanoparticles is achieved by using aseptic technique

throughout their preparation & processing & formulation & by sterilizing
treatments like autoclaving or γ- irradiation

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CHARACTERIZATION OF NANOPARTICLES
PARAMETER CHARACTERIZATION METHOD
Particle size & size distribution Photon correlation spectroscopy, Laser
defractometry, Transmission electron

microscopy, SEM, Atomic force
microscopy, Mercury porosimetry

Charge determination Laser doppler anemometry, Zeta
potentiometer
Surface hydrophobicity Water contact angle measurements, Rose
Bengal (dye) binding X-ray photoelectron
spectroscopy
Chemical analysis of surface Static secondary ion mass spectrometry,
sorptometer
Carrier-drug interaction Differential scanning calorimetry
Nanoparticle dispersion stability Critical flocculation temp (CFT)
Release profile In vitro release characteristics under physiologic &
sink conditions
Drug stability Bioassay of drug extracted from nanoparticle5s 8
w w w .D u lo M i x .c o m Chemical analysis of drug

Evaluation parameter of Nanoparticles

1. Particle size
2. Density
3. Molecular weight
4. Structure and crystallinity
5. Specific surface area
6. Surface charge & electronic mobility
7. Surface hydrophobicity
8. In-vitro release
9. Nanoparticle yield
10.Drug entrapment efficiency

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1.PARTICLE SIZE :
• Photon correlation spectroscopy (PCS) : For smaller particle.
• Laser diffractrometry : For larger particle.
• Electron microscopy (EM) : Required coating of conductive material such
as gold & limited to dry sample.
• Transmission electron microscopy (TEM) : Easier method & Permits
differntiation among nanocapsule & nanoparticle.
• Atomic force microscope
Laser force microscope
Scanning electron microscope

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2.DENSITY :
• Helium or air using a gas pycnometer
• Density gradient centrifugation

3. MOLECULAR WEIGHT :
• Gel permeation chromatography using refractive index detector.

4. STRUCTURE & CRYSTALLINITY :
• X-ray diffraction
• Thermoanalytical method such as,
1) Differential scanning calorimetry
2) Differential thermal analysis
3) Thermogravimetry

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5. SPECIFIC SURFACE AREA :
• Sorptometer
Specific Surface Area, A = 6 / ρ.d
Where, ρ is the density & d is the diameter of the particle

6. SURFACE CHARGE & ELECTRONIC MOBILITY :
• Surface charge of particle can be determined by measuring particle velocity
in electrical field.
• Laser Doppler Anemometry tech. for determination of Nanoparticles
velocities.
• Surface charge is also measured as electrical mobility.
• Charged composition critically decides biodistribution of nanoparticle .
• Zeta potential can also be obtain by measuring the electronic mobility.

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7. SURFACE HYDROPHOBICITY :
• Important influence on interaction of nanoparticles with biological
environment.
• Several methods have been used,
1 Hydrophobic interaction chromatography.
2 Two phase partition.
3 Contact angle measurement.

8. INVITRO RELEASE :
• Diffusion cell
Recently introduce modified Ultra-filtration tech.
• Media used : phosphate buffer

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8. YIELD OF NANO PARTICLE :
The yield of nanoparticles was determined by comparing the whole weight
of nanoparticles formed against the combined weight of the copolymer and
drug.

9.DRUG ENTRAPMENT EFFICIENCY:

➢ The nanoparticles were separated from the aqueous medium by
ultracentrifugation at 10,000 rpm for 30 min.
➢ Then the resulting supernatant solution was decanted and dispersed into

phosphate buffer saline pH 7.4. 64
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➢ The amount of drug present in clear supernatant after centrifugation for
30 min at 10,000 rpm was determined by UV spectroscopy.

➢ The amount of drug in supernatant was then subtracted from the total
amount of drug added during preparation of nanoparticle (W).

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QUESTIONS :

1) Discuss about Nanoparticles. (5M)
2) Discuss the development of Nanoparticles for drug targeting. How are,
they evaluated? (20M)
3) Write a short note on Nanoparticles. (5M)
4) List out the methods to prepare nanoparticles & explain any one method.
(5M)
3) State briefly on targeted drug delivery system with special reference to

nanoparticles

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REFERENCES :

o Vyas S.P. , Khar R.K. Targeted & Controlled Drug Delivery, Novel Carrier Systems,
CBS Publication,2002, Page No.331-386.

o Google.com(images)
o Jain N. K., Controlled and novel Drug Delivery, 1st edition 2001, CBS Publication;

292 – 301.

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THANK YOU

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