Nanoparticles are nanometer-sized carriers that can be used to deliver drugs and biomolecules. They have unique properties due to their small size. Various methods are used to prepare nanoparticles including emulsion-solvent evaporation, salting out, and supercritical fluid technology. Nanoparticles are characterized based on their size, morphology, surface charge, and hydrophobicity using techniques like electron microscopy and zeta potential measurements. Particle size affects properties like drug release rate and stability of the nanoparticle dispersion.

1. Targeting MethodsTargeting Methods--NanoparticlesNanoparticles
Dr. Atish S. Mundada
Associate Professor,
SNJB’s SSDJ College of Pharmacy,
Chandwad, Dist. Nashik

2. Nanotechnology:Nanotechnology:
• Nanotechnology is science,
engineering and
technology conducted at the
nanoscale, which is about
1 to 100 nanometers.
• The Nobel prize in Physics
(1965) winner Richard
Feynman is known as the
father of nanotechnology.
• Over a decade later,
Professor Norio Taniguchi
actually coined the term
nanotechnology. Five generations of nanotechnology progressFive generations of nanotechnology progress
5th: Converging
technologies

3. • Application of Nanotechnology in different fields

4. Introduction:Introduction:
• Nanotechnology is the understanding and control of matter
generally in the 1–100nm dimension range.
• The application of nanotechnology to medicine, known as
nanomedicine, concerns the use of precisely engineered materials
at this length scale to develop novel therapeutic and diagnostic
modalities.
• Nanomaterials have unique physicochemical properties, such as
ultra small size, large surface area to mass ratio and high
reactivity, which are different from bulk materials of the same
composition.
• The use of materials in nanoscale provides unparallel freedom to
modify fundamental properties such as solubility, diffusivity,
blood circulation half-life, drug release characteristics, and
immunogenicity.
• These properties can be used to overcome some of the limitations
found in traditional therapeutic and diagnostic agents.

5. • Nanoparticle drug delivery systems are nanometeric carriers used
to deliver drugs or biomolecules.
• Generally, nanometeric carriers also comprise sub-micron particles
with size below 1000 nm and with various morphologies, including
nanospheres, nanocapsules, nano-micelles, nanoliposomes and
nanodrugs, etc.
• As drug delivery system, nanoparticles can entrap drugs or
biomolecules into their interior structures and/or absorb drugs or
biomolecules onto their exterior surfaces.
• Presently, nanoparticles have been widely used to deliver drugs,
polypeptides, proteins, vaccines, nucleic acids, genes and so on.
• Over the years, nanoparticle drug delivery systems have shown
huge potential in biological, medical and pharmaceutical
applications.

6. • The researches on nanoparticle drug delivery system focus on:
(1) the selectness and combination of carrier materials to obtain
suitable drug release speed;
(2) the surface modification of nanoparticles to improve their
targeting ability;
(3) the optimization of the preparation of nanoparticles to increase
their drug delivery capability, their application in clinics and the
possibility of industrial production;
(4) the investigation of in vivo dynamic process to disclose the
interaction of nanoparticles with blood and targeting tissues and
organs, etc.
• One type of nanoparticle, which is differentiated from any of the
above terms, is a SLN with a lipid core that is solid at RT.
• During formation of SLNs the solid lipid is first melted, then
emulsified as a liquid to form an o/w emulsion, and cooled to allow
the lipid to solidify. Due to its similarity in formation and content,
these particles are referred as ‘‘emulsions with solid fat globules’’.

7. • Nanoparticle drug delivery systems have outstanding advantages
some of which include;
(1) They can pass through the smallest capillary vessels because of
their ultra-tiny volume and avoid rapid clearance by phagocytes so
that their duration in blood stream is greatly prolonged;
(2) They can penetrate cells and tissue gap to arrive at target organs
such as liver, spleen, lung, spinal cord and lymph;
(3) They could show controlled- release properties due to the
biodegradability, pH, ion and/or temperature sensibility of
materials;
(4) They can improve the utility of drugs and reduce toxic side effects.

8. Classification of nanoparticles:Classification of nanoparticles:
• There are various approaches for classification of nanomaterials.
• Nanoparticles are classified based on one, two and three
dimensions.
One dimension nanoparticles:
• One dimensional system, such as thin film or manufactured
surfaces, has been used for decades in electronics, chemistry and
engineering.
• Production of thin films (sizes1-100 nm) or monolayer is now
common place in the field of solar cells or catalysis.
• This thin films are using in different technological applications,
including information storage systems, chemical and biological
sensors, fibre-optic systems, magneto-optic and optical device.

9. Two dimension nanoparticles:
• Carbon nanotubes (CNTs):
• Carbon nanotubes are hexagonal network of carbon atoms, 1 nm in
diameter and 100 nm in length, as a layer of graphite rolled up into
cylinder.
• CNTs are of two types, single walled carbon nanotubes (SWCNTs)
and multi-walled carbon nanotubes (MWCNTs).
• The small dimensions of carbon nanotubes, combined with their
remarkable physical, mechanical and electrical properties, make
them unique materials.
• The mechanical strength of carbon nanotubes is sixty times greater
than the best steels.
• Carbon nanotubes have a great capacity for molecular absorption
and offering a three dimensional configuration.
• Moreover they are chemically and chemically very stable.

10. Three dimension nanoparticles:
• Fullerenes (Carbon 60): This is a hollow ball composed of
interconnected carbon pentagons and hexagons, resembling a
soccer ball.
• Fullerenes are class of materials displaying unique physical
properties.
• Since fullerenes are empty structures with dimensions similar to
several biological active molecules, they can be filled with
different substances and find potential medical applications.
• Dendrimers: Dendrimers represents a new class of controlled-
structure polymers with nanometric dimensions.
• Dendrimers used in drug delivery and imaging are usually 10 to
100 nm in diameter with multiple functional groups on their
surface, rendering them ideal carriers for targeted drug delivery.

11. • Quantum Dots (QDs): Quantum dots are small devices that
contain a tiny droplet of free electrons.
• QDs are colloidal semiconductor nanocrystals ranging from 2 to 10
nm in diameter. QDs can be synthesized from various types of
semiconductor materials via colloidal synthesis or
electrochemistry.
• The most commonly used QDs are cadmium selenide (CdSe),
cadmium telluride (CdTe), indium phosphide (InP), and indium
arsenide (InAs).
• QDs can have anything from a single electron to a collection of
several thousands. The size, shape and number of electrons can be
precisely controlled. They have been developed in a form of
semiconductors, insulators, metals, magnetic materials or metallic
oxides.
• QDs also provide enough surface area to attach therapeutic agents
for simultaneous drug delivery and in vivo imaging, as well as for
tissue engineering.

12. Preparation of nanoparticles:Preparation of nanoparticles:
• The selection of appropriate method for the preparation of
nanoparticles depends on the physicochemical character of the
polymer and the drug to be loaded.
• The primary manufacturing methods of nanoparticles from
preformed polymer includes:
– Emulsion-Solvent Evaporation Method
• Double Emulsion and Evaporation Method
– Salting Out Method
» Emulsions- Diffusion Method
• Solvent Displacement / Precipitation method
• Supercritical Fluid Technology

13. 1. Emulsion-Solvent Evaporation Method:
• This is one of the most frequently used methods for the preparation
of nanoparticles.
• Emulsification-solvent evaporation involves two steps.
– The first step requires emulsification of the polymer solution
into an aqueous phase.
– During the second step polymer solvent is evaporated, inducing
polymer precipitation as nanospheres.
• The nano particles are collected by ultracentrifugation and washed
with distilled water to remove stabilizer residue or any free drug
and lyophilized for storage.

14. • Modification of this method is known as high pressure
emulsification and solvent evaporation method.
• This method involves preparation of a emulsion which is then
subjected to homogenization under high pressure followed by
overall stirring to remove organic solvent.
• The size can be controlled by adjusting the stirring rate, type and
amount of dispersing agent, viscosity of organic and aqueous
phases and temperature.
• However this method can be applied to liposoluble drugs and
limitation are imposed by the scale up issue.
• Polymers used in this method are PLA, PLGA, Ethyl Cellulose,
cellulose acetate phthalate, Poly ε-caprolactone) (PCL), Poly (β-
hydroxybutyrate) (PHB).

15. 2. Double Emulsion and Evaporation Method:
• The emulsion and evaporation method suffer from the limitation
of poor entrapment of hydrophilic drugs.
• Therefore to encapsulate hydrophilic drug the double emulsion
technique is employed, which involves the addition of aqueous
drug solutions to organic polymer solution under vigorous stirring
to form w/o emulsions.
• This w/o emulsion is added into second aqueous phase with
continuous stirring to form the w/o/w emulsion.
• The emulsion then subjected to solvent removal by evaporation
and nano particles can be isolated by centrifugation at high speed.
• In this method the amount of hydrophilic drug to be incorporated,
the concentration of stabilizer used, the polymer concentration, the
volume of aqueous phase are the variables that affect the
characterization of nanoparticles.

16. 3. Salting Out Method:
• 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 poly vinyl pyrrolidone or hydroxy ethyl cellulose.
• This oil/water 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.

17. • Several manufacturing parameters can be varied including stirring
rate, internal/external phase ratio, concentration of polymers in the
organic phase, type of electrolyte concentration and type of
stabilizer in the aqueous phase.
• This technique used in the preparation of PLA, Poly( methacrylic)
acids, and Ethyl cellulose nanospheres leads to high efficiency and
is easily scaled up.
• Salting out does not require an increase of temperature and therefore
may be useful when heat sensitive substances have to be processed.
• The greatest disadvantages are exclusive application to lipophilic
drug and the extensive nanoparticles washing steps.

18. 4. Emulsions- Diffusion Method:
• This is another widely used method to prepare nanoparticles. The
encapsulating polymer is dissolved in a partially water-miscible
solvent (such as propylene carbonate, benzyl alcohol) and saturated
with water to ensure the initial thermodynamic equilibrium of both
liquids.
• Subsequently, the polymer-water saturated solvent phase is
emulsified in an aqueous solution containing stabilizer, leading to
solvent diffusion to the external phase and the formation of
nanospheres or nanocapsules, according to the oil-to-polymer ratio.
• Finally, the solvent is eliminated by evaporation or filtration,
according to its boiling point.

19. • This technique presents several advantages, such as high
encapsulation efficiencies (generally 70%), no need for
homogenization, high batch-to-batch reproducibility, ease of scale
up, simplicity, and narrow size distribution.
• Disadvantages are the high volumes of water to be eliminated
from the suspension and the leakage of water-soluble drug into the
saturated-aqueous external phase during emulsification, reducing
encapsulation efficiency.
• Several drug- loaded nano particles were produced by the
technique, including mesotetra (hydroxyphenyl) porphyrin-loaded
PLGA (p-THPP) nano particles, doxorubicin-loaded PLGA nano
particles and cyclosporine loaded sodium glycolate nanoparticles.

20. 5. Solvent Displacement / Precipitation method:
• Solvent displacement involves the precipitation of a preformed
polymer from an organic solution and the diffusion of the organic
solvent in the aqueous medium in the presence or absence of
surfactant.
• Polymers, drug, and or lipophilic surfactant are dissolved in a
semipolar water miscible solvent such as acetone or ethanol.
• The solution is then poured or injected into an aqueous solution
containing stabilizer under magnetic stirring.
• Nano particles are formed instantaneously by the rapid solvent
diffusion. The solvent is then removed from the suspensions under
reduced pressure.

21. • The rates of addition of the organic phase into the aqueous phase affect
the particles size.
• It was observed that a decrease in both particles size and drug
entrapment occurs as the rate of mixing of the two phase increases.
• Nano precipitation method is well suited for most of the poorly soluble
drugs.
• Nanospheres size, drug release and yield were shown to be effectively
controlled by adjusting preparation parameters.
• Adjusting polymer concentration in the organic phase was found to be
useful in the production of smaller sized nanospheres through restricted
to a limited range of the polymer to drug ratio

22. 6. Supercritical fluid technology
• Supercritical fluid technology method is alternative method
because in this method organic solvents are not used which are
hazardous to the environment as well as to physiological systems.
• Supercritical fluid is defined as a solvent at a temperature above
its critical temperature at which the fluid remains a single phase
regardless of pressure.
• Supercritical CO2 is the most widely used supercritical fluid
because of its mild critical conditions (Tc = 31.1 °C, Pc = 73.8
bars) its nontoxicity, non-flammability and low price.
• Mainly supercritical fluid used in two main techniques:
– Supercritical anti-solvent (SAS)
– Rapid expansion of critical solution (RESS).

23. • In SAS process liquid solvents are used, which should completely
miscible with the supercritical fluid.
• The process of SAS employs a liquid solvent, eg methanol, which
is completely miscible with the supercritical fluid, the extract of
the liquid solvent by supercritical fluid leads to the instantaneous
precipitation of the solute, it results the formation of nanoparticles.
• In RESS high degree of super saturation occurs by dissolving
solute in a supercritical fluid to form a solution, followed by the
rapid expansion of the solution across an orifice or a capillary
nozzle into ambient air by the rapid pressure reduction in the
expansion which results in homogenous nucleation and thereby,
the formation of well-dispersed particles.

24. Characterization of Nanoparticles:Characterization of Nanoparticles:
• Nanoparticles are generally characterized by their size,
morphology and surface charge, using advanced microscopic
techniques such as scanning electron microscopy (SEM),
transmission electron microscopy (TEM) and atomic force
microscopy (AFM).
• The average particle diameter, their size distribution and charge
affect the physical stability and the in vivo distribution of the
nanoparticles.
• Electron microscopy techniques are very useful in ascertaining the
overall shape of polymeric nanoparticles, which may determine
their toxicity.
• The surface charge of the nanoparticles affects the physical
stability and redispersibility of the polymer dispersion as well as
their in vivo performance.

25. • Particle size:
• Particle size distribution and morphology are the most important
parameters of characterization of nanoparticles.
• Morphology and size are measured by electron microscopy.
• The major application of nanoparticles is in drug release and drug
targeting. It has been found that particle size affects the drug
release.
• Smaller particles offer larger surface area. As a result, most of the
drug loaded onto them will be exposed to the solvent leading to
fast drug release.
• On the contrary, drugs slowly diffuse inside larger particles. Even
smaller particles tend to aggregate during storage and
transportation of nanoparticle dispersion. Hence, there is a
compromise between a small size and maximum stability of
nanoparticles.
• Polymer degradation can also be affected by the particle size. For
instance, the degradation rate of poly (lactic-co-glycolic acid) was
found to increase with increasing particle size in vitro.

26. • Surface Charge:
• The nature and intensity of the surface charge of nanoparticles is
very important as it determines their interaction with the biological
environment as well as their electrostatic interaction with
bioactive compounds.
• The colloidal stability is analyzed through zeta potential of
nanoparticles.
• This potential is an indirect measure of the surface charge. It
corresponds to potential difference between the outer Helmholtz
plane and the surface of shear.
• The measurement of the zeta potential allows for predictions
about the storage stability of colloidal dispersion.
• High zeta potential values, either positive or negative, should be
achieved in order to ensure stability and avoid aggregation of the
particles.

27. • Surface hydrophobicity:
• Surface hydrophobicity can be determined by several techniques
such as hydrophobic interaction chromatography, biphasic
partitioning, adsorption of probes, contact angle measurements
etc.
• Recently, several sophisticated analytical techniques are reported
in literature for surface analysis of nanoparticles.
• X – ray photon correlation spectroscopy permits the identification
of specific chemical groups on the surface of nanoparticles.
• The extent of surface hydrophobicity can be predicted from the
values of zeta potential.

28. • Drug Release:
• A central reason for pursuing nanotechnology is to deliver drugs,
hence understanding the manner and extent to which the drug
molecules are released is important.
• In order to obtain such information most release methods require
that the drug and its delivery vehicle be separated.
• The drug loading of the nanoparticles is generally defined as the
amount of drug bound per mass of polymer (usually moles of drug
per mg polymer or mg drug per mg polymer); it could also be
given as percentage relative to the polymer.
• The technique used for this analysis is classical analytical methods
like UV spectroscopy or high performance liquid chromatography
(HPLC) after ultracentrifugation, ultra filtration, gel filtration, or
centrifugal ultrafiltration.
• Quantification is performed with the UV spectroscopy or HPLC.

29. • Different parameters and characterization methods for nanoparticles

30. NanotechnologyNanotechnology--based drug delivery systems:based drug delivery systems:
Smart drug delivery systems:-
• Ideally, nanoparticulate drug delivery system should selectively
accumulate in the required organ or tissue and at the same time,
penetrate target cells to deliver the bioactive agent.
• It has been suggested that, organ or tissue accumulation could be
achieved by the passive or antibody-mediated active targeting,
while the intracellular delivery could be mediated by certain
ligands or by cell-penetrating peptides.
• Thus, a drug delivery system (DDS) should be multifunctional and
possess the ability to switch on and switch off certain functions
when necessary.
• Another important requirement is that different properties of the
multifunctional DDS are coordinated in an optimal fashion.
• Nanoparticulate DDS, such as liposomes and micelles, are
frequently used to increase the efficacy of drug and DNA delivery
and targeting.

31. • The development of a DDS built in such a way that during the first
phase of delivery, a nonspecific cell-penetrating function is
shielded by the organ/tissue-specific delivery will be possible.
• Upon accumulating in the target, protecting polymer or antibody
attached to the surface of the DDS via the stimuli sensitive bond
should detach under the action of local pathological conditions
such as abnormal pH or temperature and expose the previously
hidden second function allowing for the subsequent delivery of the
carrier and its cargo inside cells.
• While such DDS should be stable in the blood for a long time to
allow for an efficient target accumulation, it has to lose the
protective coat inside the target almost instantly to allow for fast
internalization thereby minimizing the washing away of the
released drug or DNA.
• Intracellular trafficking, distribution, and fate of the carrier and its
cargo can be additionally controlled by its charge and
composition.

32. • One of the most outstanding achievements in the drug delivery
field was the development of smart drug delivery systems
(SDDSs), also called stimuli-sensitive delivery systems.
• SDDS provides a programmable and predictable drug release
profile in response to various stimulation sources.
• The conventional controlled release systems are based on the
predetermined drug release rate irrespective of the environmental
condition at the time of application. On the other hand, SDDS is
based on the release-on-demand strategy, allowing a drug carrier to
liberate a therapeutic drug only when it is required in response to a
specific stimulation.
• The best example of SDDS has been self-regulated insulin delivery
systems that can respond to changes in the environmental glucose
level.

33. • One of the most widely used SDDSs has been polymeric micelles.
• Many polymeric micelles consisting of hydrophobic and
hydrophilic polymer blocks have been developed.
• They have been found to dissolve water-insoluble drugs, such as
doxorubicin or paclitaxel, at high concentrations.
• When administered to the body, drug release from polymeric
micelles usually depends on simple diffusion, degradation of the
micelle blocks, or disruption of the micelles by body components.
• The release kinetics of the loaded drug can be modulated by
varying the degradation rate of hydrophobic polymer blocks, but
because the degradation rate is usually very slow, the loaded drug
is released by diffusion from polymeric micelles.

34. Polymer–drug conjugates:-
• Polymer–drug conjugates are a class of polymer therapeutics that
consists of a water-soluble polymer that is chemically conjugated
to a drug through a biodegradable linker.
• The idea started in 1975 when Ringsdorf proposed the use of
polymer–drug conjugates to deliver hydrophobic small molecules.
• The reasoning was that, small molecule drugs, especially
hydrophobic compounds, have a low aqueous solubility and a
broad tissue distribution profile such that, administration of the free
drug may result in serious side effects.
• Therefore, conjugation of these compounds to hydrophilic,
biocompatible polymers would significantly increase their aqueous
solubility, modify their tissue distribution profile and enhance their
plasma circulation half-life.

35. • Over the last decade, polymer conjugate technology has proven to
be a viable formulation strategy. There have been reports of bio-
conjugation of protein and peptide to PEG been able to
significantly improve the efficacy of these macro- molecular drugs
by increasing their stability in the presence of proteases and
decreasing their immunogenicity.
• Studies have also shown that by using PEG in a specific molecular
weight range, the fast renal clearance and mononuclear phagocytic
system uptake of the drugs can be prevented or delayed leading to
a prolonged plasma half-life for the conjugated molecules.
Successful applications have led to several FDA- approved
products e.g. Neulasta®.
• The first practical use of polymer therapeutics that resulted in an
FDA-approved anti-cancer treatment was the introduction of PEG-
Lasparaginase (Oncaspar1) in 1994.

36. • One of the major advantages of polymer–drug conjugates is
prolonged circulation in the blood stream by retarding
degradation/ metabolism/excretion rates of the conjugated drugs.
• Many peptide and protein drugs cannot be delivered by oral
administration because of their large molecular weights.
• Even when administered directly into the blood stream, they do not
remain in the blood for a long time due to fast degradation and
metabolism, limiting the clinical applications.
• The circulation times of these drugs have increased substantially
by conjugation with polymers.
• Very often, low molecular weight drugs with high hydrophobicity
are used for conjugation with attendant reduction in the
degradation/clearance rate as well as the toxicity of the conjugated
drug.
• The polymers used in conjugation usually have stimuli-
responsiveness, imparting unique properties into the conjugated
drug such that, its activities can be turned on or off by external
signals.

37. Multifunctional drug carriers:-
• A multifunctional drug delivery system (MDDS) refers to drug
carrier that has multiple properties of prolonged blood circulation,
passive or active localization at specific disease site, stimuli-
sensitivity, ability to deliver drug into intracellular target
organelles, and/or imaging ability.
• Some reported MDDS include the biotin-tagged pH-sensitive
polymeric micelles based on a mixture of PLA-b-PEG-b-PHis-
biotin (PLA=poly (L-lactic acid)) and PEG-b-PHis block
copolymers by Lee et al in which the targeting moiety, biotin, was
masked until the carrier was exposed to an expected environment
of pH 7.0.
• Once the nanocarrier was internalized to cancer cells by ligand–
receptor interactions, lowered pH (< 6.5) destabilized the carrier
resulting in a burst release of the loaded drug.

38. Organic/inorganic composites:-
• An inorganic-organic composite usually comprises an inorganic
phase and a film forming organic phase.
• A typical green approach to developing an inorganic-organic
composite involves the selection of film forming organic phase
from starches having a degree of polymerization; degree of
substitution and viscosity such that the substituted starches are
insoluble in water during mixing but dissolve at a higher
processing temperature during forming, setting or drying of the
composite.
• Thus, excessive migration of the starch is prevented and the
composite is substantially strengthened.
• Recently, combined with other technologies such as optics, single
molecular imaging, or cell/protein-based assay systems,
biomedical lab-on-a-chip devices have become an important part
of drug discovery and diagnosis.

39. • List of Marketed Nanoparticle Products