Mohanraj & Chen
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Tropical Journal of Pharmaceutical Research, June 2006; 5 (1): 561-573
© Pharmacotherapy Group,
Faculty of Pharmacy, University of Benin,
Benin City, Nigeria.
All rights reserved.
Available online at http://www.tjpr.freehosting.net
Research Article
Nanoparticles – A Review
VJ Mohanraj
1*
and Y Chen
2
1Orchid Chemicals & Pharmaceuticals Limited, Chennai, India
2 School of Pharmacy, Curtin University of Technology, Perth, Australia
Abstract
For the past few decades, there has been a considerable research interest in the area of drug
delivery using particulate delivery systems as carriers for small and large molecules.
Particulate systems like nanoparticles have been used as a physical approach to alter and
improve the pharmacokinetic and pharmacodynamic properties of various types of drug
molecules. They have been used in vivo to protect the drug entity in the systemic circulation,
restrict access of the drug to the chosen sites and to deliver the drug at a controlled and
sustained rate to the site of action. Various polymers have been used in the formulation of
nanoparticles for drug delivery research to increase therapeutic benefit, while minimizing side
effects. Here, we review various aspects of nanoparticle formulation, characterization, effect
of their characteristics and their applications in delivery of drug molecules and therapeutic
genes.
Key words: nanoparticles, drug delivery, targeting, drug release
*Corresponding Author: E-mail: mohanraj_67@hotmail.com, Tel: +91-9840464216
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INTRODUCTION
Nanoparticles are defined as particulate
dispersions or solid particles with a size in the
range of 10-1000nm. The drug is dissolved,
entrapped, encapsulated or attached to a
nanoparticle matrix. Depending upon the method
of preparation, nanoparticles, nanospheres or
nanocapsules can be obtained. Nanocapsules
are systems in which the drug is confined to a
cavity surrounded by a unique polymer
membrane, while nanospheres are matrix
systems in which the drug is physically and
uniformly dispersed. In recent years,
biodegradable
polymeric
nanoparticles,
particularly those coated with hydrophilic
polymer such as poly(ethylene glycol) (PEG)
known as long-circulating particles, have been
used as potential drug delivery devices because
of their ability to circulate for a prolonged period
time target a particular organ, as carriers of DNA
in gene therapy, and their ability to deliver
proteins, peptides and genes 1-4.
The major goals in designing nanoparticles as a
delivery system are to control particle size,
surface
properties
and
release
of
pharmacologically active agents in order to
achieve the site-specific action of the drug at the
therapeutically optimal rate and dose regimen.
Though liposomes have been used as potential
carriers with unique advantages including
protecting drugs from degradation, targeting to
site of action and reduction toxicity or side
effects, their applications are limited due to
inherent problems such as low encapsulation
efficiency, rapid leakage of water-soluble drug in
the presence of blood components and poor
storage stability. On the other hand, polymeric
nanoparticles offer some specific advantages
over liposomes. For instance, they help to
increase the stability of drugs/proteins and
possess useful controlled release properties 5, 6.
The advantages of using nanoparticles as a drug
delivery system include the following:
1. Particle size and surface characteristics of
nanoparticles can be easily manipulated to
achieve both passive and active drug targeting
after parenteral administration.
2. They control and sustain release of the drug
during the transportation and at the site of
localization, altering organ distribution of the
drug and subsequent clearance of the drug so
as to achieve increase in drug therapeutic
efficacy and reduction in side effects.
3. Controlled release and particle degradation
characteristics can be readily modulated by the
choice of matrix constituents.
Drug loading is relatively high and drugs can be
incorporated into the systems without any
chemical reaction; this is an important factor for
preserving the drug activity.
4. Site-specific targeting can be achieved by
attaching targeting ligands to surface of particles
or use of magnetic guidance.
5. The system can be used for various routes of
administration including oral, nasal, parenteral,
intra-ocular etc.
In spite of these advantages, nanoparticles do
have limitations. For example, their small size
and large surface area can lead to particle-
particle aggregation, making physical handling of
nanoparticles difficult in liquid and dry forms. In
addition, small particles size and large surface
area readily result in limited drug loading and
burst release. These practical problems have to
be overcome before nanoparticles can be used
clinically or made commercially available. The
present review details the latest development of
nanoparticulate drug delivery systems, surface
modification issues, drug loading strategies,
release control and potential applications of
nanoparticles.
Preparation of Nanoparticles
Nanoparticles can be prepared from a variety of
materials such as proteins, polysaccharides and
synthetic polymers. The selection of matrix
materials is dependent on many factors
including7: (a) size of nanoparticles required; (b)
inherent properties of the drug, e.g., aqueous
solubility and stability; (c) surface characteristics
such as charge and permeability; (d) degree of
biodegradability, biocompatibility and toxicity; (e)
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Drug release profile desired; and (f) Antigenicity
of the final product.
Nanoparticles have been prepared most
frequency by three methods: (1) dispersion of
preformed polymers; (2) polymerization of
monomers; and (3) ionic gelation or coacervation
of hydrophilic polymers. However, other methods
such as supercritical fluid technology 8
and
particle replication in non-wetting templates
(PRINT) 9
have also been described in the
literature for production of nanoparticles. The
latter was claimed to have absolute control of
particle size, shape and composition, which
could set an example for the future mass
production of nanoparticles in industry.
Dispersion of preformed polymers: Dispersion of
preformed polymers is a common technique
used to prepare biodegradable nanoparticles
from poly (lactic acid) (PLA); poly (D,L-glycolide),
PLG; poly (D, L-lactide-co-glycolide) (PLGA) and
poly (cyanoacrylate) (PCA), 10-12. This technique
can be used in various ways as described below.
Solvent evaporation method: In this method, the
polymer is dissolved in an organic solvent such
as dichloromethane, chloroform or ethyl acetate
which is also used as the solvent for dissolving
the hydrophobic drug. The mixture of polymer
and drug solution is then emulsified in an
aqueous solution containing a surfactant or
emulsifying agent to form an oil in water (o/w)
emulsion. After the formation of stable emulsion,
the organic solvent is evaporated either by
reducing the pressure or by continuous stirring.
Particle size was found to be influenced by the
type and concentrations of stabilizer,
homogenizer speed and polymer concentration
13. In order to produce small particle size, often a
high-speed homogenization or ultrasonication
may be employed
14.
Spontaneous emulsification or solvent diffusion
method:This is a modified version of solvent
evaporation method 15. In this method, the water-
miscible solvent along with a small amount of the
water immiscible organic solvent is used as an
oil phase. Due to the spontaneous diffusion of
solvents an interfacial turbulence is created
between the two phases leading to the formation
of small particles. As the concentration of water
miscible solvent increases, a decrease in the
size of particle can be achieved.
Both solvent evaporation and solvent diffusion
methods can be used for hydrophobic or
hydrophilic drugs. In the case of hydrophilic
drug, a multiple w/o/w emulsion needs to be
formed with the drug dissolved in the internal
aqueous phase.
Polymerization method
In this method, monomers are polymerized to
form nanoparticles in an aqueous solution . Drug
is incorporated either by being dissolved in the
polymerization medium or by adsorption onto the
nanoparticles after polymerization completed.
The nanoparticle suspension is then purified to
remove various stabilizers and surfactants
employed for polymerization by ultra-
centrifugation and re-suspending the particles in
an isotonic surfactant-free medium. This
technique has been reported for making
polybutylcyanoacrylate
or
poly
(alkylcyanoacrylate)
nanoparticles16;17.
Nanocapsule formation and their particle size
depends on the concentration of the surfactants
and stabilizers used 18.
Coacervation or ionic gelation method
Much research has been focused on the
preparation of nanoparticles using biodegradable
hydrophilic polymers such as chitosan, gelatin
and sodium alginate. Calvo and co-workers
developed a method for preparing hydrophilic
chitosan nanoparticles by ionic gelation 19, 20.
The method involves a mixture of two aqueous
phases, of which one is the polymer chitosan, a
di-block co-polymer ethylene oxide or propylene
oxide (PEO-PPO) and the other is a polyanion
sodium tripolyphosphate. In this method,
positively charged amino group of chitosan
interacts with negative charged tripolyphosphate
to form coacervates with a size in the range of
nanometer. Coacervates are formed as a result
of electrostatic interaction between two aqueous
phases, whereas, ionic gelation involves the
material undergoing transition from liquid to gel
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564
due to ionic interaction conditions at room
temperature.
Production
of
nanoparticles
using
supercritical fluid technology
Conventional methods such as solvent
extraction-evaporation, solvent diffusion and
organic phase separation methods require the
use of organic solvents which are hazardous to
the environment as well as to physiological
systems. Therefore, the supercritical fluid
technology has been investigated as an
alternative to prepare biodegradable micro- and
nanoparticles because supercritical fluids are
environmentally safe 21.
A supercritical fluid can be generally defined as
a solvent at a temperature above its critical
temperature, at which the fluid remains a single
phase regardless of pressure 21. Supercritical
CO2
(SC CO2) is the most widely used
supercritical fluid because of its mild critical
conditions (
Tc = 31.1 °C,
Pc = 73.8 bars), non-
toxicity, non-flammability, and low price. The
most common processing techniques involving
supercritical fluids are supercritical anti-solvent
(SAS) and rapid expansion of critical solution
(RESS). The process of SAS employs a liquid
solvent, eg methanol, which is completely
miscible with the supercritical fluid (SC CO2), to
dissolve the solute to be micronized; at the
process conditions, because the solute is
insoluble in the supercritical fluid, the extract of
the liquid solvent by supercritical fluid leads to
the instantaneous precipitation of the solute,
resulting the formation of nanoparticles 8. Thote
and Gupta (2005) reported the use of a modified
SAS method for formation of hydrophilic drug
dexamethasone phosphate drug nanoparticles
for microencapsulation purpose 22.
RESS differs from the SAS process in that its
solute is dissolved in a supercritical fluid (such
as supercritical methanol) and then the solution
is rapidly expanded through a small nozzle into a
region lower pressure 21 , Thus the solvent
power of supercritical fluids dramatically
decreases and the solute eventually precipitates.
This technique is clean because the precipitate
is basically solvent free. RESS and its modified
process have been used for the product of
polymeric nanoparticles 23. Supercritical fluid
technology technique, although environmentally
friendly and suitable for mass production,
requires specially designed equipment and is
more expensive.
Effect of Characteristics of Nanoparticles on
Drug Delivery
Particle size
Particle size and size distribution are the most
important characteristics of nanoparticle
systems. They determine the
in vivo distribution,
biological fate, toxicity and the targeting ability of
nanoparticle systems. In addition, they can also
influence the drug loading, drug release and
stability of nanoparticles.
Many studies have demonstrated that
nanoparticles of sub-micron size have a number
of advantages over microparticles as a drug
delivery system 24. Generally nanoparticles have
relatively higher intracellular uptake compared to
microparticles and available to a wider range of
biological targets due to their small size and
relative mobility. Desai et al found that 100 nm
nanoparticles had a 2.5 fold greater uptake than
1 µm microparticles, and 6 fold greater uptake
than 10 µm microparticles in a Caco-2 cell line25.
In a subsequent study 26, the nanoparticles
penetrated throughout the submucosal layers in
a rat in situ intestinal loop model, while
microparticles were predominantly localized in
the epithelial lining. It was also reported that
nanoparticles can across the blood-brain barrier
following the opening of tight junctions by hyper
osmotic mannitol, which may provide sustained
delivery of therapeutic agents for difficult-to-treat
diseases like brain tumors 27. Tween 80 coated
nanoparticles have been shown to cross the
blood-brain barrier 28. In some cell lines, only
submicron nanoparticles can be taken up
efficiently but not the larger size microparticles
29.
Drug release is affected by particle size. Smaller
particles have larger surface area, therefore,
most of the drug associated would be at or near
the particle surface, leading to fast drug release.
Whereas, larger particles have large cores which
allow more drug to be encapsulated and slowly
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565
diffuse out 30. Smaller particles also have
greater risk of aggregation of particles during
storage and transportation of nanoparticle
dispersion. It is always a challenge to formulate
nanoparticles with the smallest size possible but
maximum stability.
Polymer degradation can also be affected by the
particle size. For instance, the rate of PLGA
polymer degradation was found to increase with
increasing particle size
in vitro 31. It was thought
that in smaller particles, degradation products of
PLGA formed can diffuse out of the particles
easily while in large particles, degradation
products are more likely remained within the
polymer matrix for a longer period to cause
autocatalytic degradation of the polymer
material. Therefore, it was hypothesized that
larger particles will contribute to faster polymer
degradation as well as the drug release.
However, Panyam et al prepared PLGA particles
with different size ranges and found that the
polymer degradation rates
in vitro were not
substantially different for different size particles
32.
Currently, the fastest and most routine method of
determining particle size is by photon-correlation
spectroscopy or dynamic light scattering.
Photon-correlation spectroscopy requires the
viscosity of the medium to be known and
determines the diameter of the particle by
Brownian motion and light scattering properties
33. The results obtained by photon-correlation
spectroscopy are usually verified by scanning or
transmission electron microscopy (SEM or
TEM).
Surface properties of nanoparticles
When
nanoparticles
are
administered
intravenously, they are easily recognized by the
body immune systems, and are then cleared by
phagocytes from the circulation 34. Apart from
the size of nanoparticles, their surface
hydrophobicity determines the amount of
adsorbed blood components, mainly proteins
(opsonins). This in turn influences the
in vivo fate
of nanoparticles 34, 35. Binding of these opsonins
onto the surface of nanoparticles called
opsonization acts as a bridge between
nanoparticles and phagocytes. The association
of a drug to conventional carriers leads to
modification of the drug biodistribution profile, as
it is mainly delivered to the mononuclear
phagocytes system (MPS) such as liver, spleen,
lungs and bone marrow. Indeed, once in the
blood
stream,
surface
non-modified
nanoparticles (conventional nanoparticles) are
rapidly opsonized and massively cleared by the
macrophages of MPS rich organs 36. Generally,
it is IgG, compliment C3 components that are
used for recognition of foreign substances,
especially foreign macromolecules.
Hence, to increase the likelihood of the success
in drug targeting by nanoparticles, it is necessary
to minimize the opsonization and to prolong the
circulation of nanoparticles
in vivo. This can be
achieved by (a) surface coating of nanoparticles
with hydrophilic polymers/surfactants; (b)
formulation of nanoparticles with biodegradable
copolymers with hydrophilic segments such as
polyethylene glycol (PEG), polyethylene oxide,
polyoxamer, poloxamine and polysorbate 80
(Tween 80).
Studies show that PEG conformation at the
nanoparticle surface is of utmost importance for
the opsonin repelling function of the PEG layer.
PEG surfaces in brush-like and intermediate
configurations reduced phagocytosis and
complement activation whereas PEG surfaces in
mushroom-like configuration were potent
complement
activators
and
favoured
phagocytosis 2, 37.
The zeta potential of a nanoparticle is commonly
used to characterise the surface charge property
of nanoparticles 38. It reflects the electrical
potential of particles and is influenced by the
composition of the particle and the medium in
which it is dispersed. Nanoparticles with a zeta
potential above (+/-) 30 mV have been shown to
be stable in suspension, as the surface charge
prevents aggregation of the particles. The zeta
potential can also be used to determine whether
a charged active material is encapsulated within
the centre of the nanocapsule or adsorbed onto
the surface.
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566
Drug loading
Ideally, a successful nanoparticulate system
should have a high drug-loading capacity
thereby reduce the quantity of matrix materials
for administration. Drug loading can be done by
two methods:
•
Incorporating at the time of
nanoparticles production (incorporation method)
•
Absorbing the drug after formation of
nanoparticles by incubating the carrier with a
concentrated drug solution (adsorption
/absorption technique).
Drug loading and entrapment efficiency very
much depend on the solid-state drug solubility in
matrix material or polymer (solid dissolution or
dispersion), which is related to the polymer
composition, the molecular weight, the drug
polymer interaction and the presence of end-
functional groups (ester or carboxyl) 39 40 41. The
PEG moiety has no or little effect on drug
loading 42. The macromolecule or protein shows
greatest loading efficiency when it is loaded at or
near its isoelectric point when it has minimum
solubility and maximum adsorption 19 For small
molecules, studies show the use of ionic
interaction between the drug and matrix
materials can be a very effective way to increase
the drug loading 43, 44.
Drug release
To develop a successful nanoparticulate system,
both drug release and polymer biodegradation
are important consideration factors. In general,
drug release rate depends on: (1) solubility of
drug; (2) desorption of the surface-
bound/adsorbed drug; (3) drug diffusion through
the nanoparticle matrix; (4) nanoparticle matrix
erosion/degradation; and (5) combination of
erosion/diffusion process. Thus solubility,
diffusion and biodegradation of the matrix
materials govern the release process.
In the case of nanospheres, where the drug is
uniformly distributed, the release occurs by
diffusion or erosion of the matrix under sink
conditions. If the diffusion of the drug is faster
than matrix erosion, the mechanism of release is
largely controlled by a diffusion process. The
rapid initial release or ‘burst’ is mainly attributed
to weakly bound or adsorbed drug to the large
surface of nanoparticles 45. It is evident that the
method of incorporation has an effect on release
profile. If the drug is loaded by incorporation
method, the system has a relatively small burst
effect and better sustained release
characteristics 46. If the nanoparticle is coated by
polymer, the release is then controlled by
diffusion of the drug from the core across the
polymeric membrane. The membrane coating
acts as a barrier to release, therefore, the
solubility and diffusivity of drug in polymer
membrane becomes determining factor in drug
release. Furthermore release rate can also be
affected by ionic interaction between the drug
and addition of auxillary ingredients. When the
drug is involved in interaction with auxillary
ingredients to form a less water soluble complex,
then the drug release can be very slow with
almost no burst release effect 43; whereas if the
addition of auxillary ingredients e.g., addition of
ethylene oxide-propylene oxide block copolymer
(PEO-PPO) to chitosan, reduces the interaction
of the model drug bovine serum albumin (BSA)
with the matrix material (chitosan) due to
competitive electrostatic interaction of PEO-PPO
with chitosan, then an increase in drug release
could be observed 20.
Various methods which can be used to study the
in vitro release of the drug are: (1) side-by-side
diffusion cells with artificial or biological
membranes; (2) dialysis bag diffusion technique;
(3) reverse dialysis bag technique; (4) agitation
followed by ultracentrifugation/centrifugation; (5)
Ultra-filtration or centrifugal ultra-filtration
techniques. Usually the release study is carried
out by controlled agitation followed by
centrifugation. Due to the time-consuming nature
and technical difficulties encountered in the
separation of nanoparticles from release media,
the dialysis technique is generally preferred.
Applications of Nanoparticulate Delivery
Systems
Tumor targeting using nanoparticulate delivery
systems
The rationale of using nanoparticles for tumor
targeting is based on 1) nanoparticles will be
able to deliver a concentrate dose of drug in the
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Trop J Pharm Res, June 2006; 5 (1)
567
vicinity of the tumor targets via the enhanced
permeability and retention effect or active
targeting by ligands on the surface of
nanoparticles; 2) nanoparticles will reduce the
drug exposure of health tissues by limiting drug
distribution to target organ.
Verdun et al demonstrated in mice treated with
doxorubicin
incorporated
into
poly
(isohexylcyanoacrylate) nanopsheres that higher
concentrations of doxorubicin manifested in the
liver, spleen and lungs than in mice treated with
free doxorubicin 47. Studies show that the
polymeric composition of nanoparticles such as
type, hydrophobicity and biodegradation profile
of the polymer along with the associated drug’s
molecular weight, its localization in the
nanospheres and mode of incorporation
technique, adsorption or incorporation, have a
great influence on the drug distribution pattern
in
vivo. The exact underlying mechanism is not
fully understood but the biodistribution of
nanoparticles is rapid, within ½ hour to 3 hours,
and
it
likely
involves
MPS
and
endocytosis/phagocytosis process 48.
Recently Bibby et al reported the biodistribution
and pharmacokinetics (PK) of a cyclic RGD-
doxorubicin-nanoparticle formulation in tumor-
bearing mice 49. Their biodistribution studies
revealed decreasing drug concentrations over
time in the heart, lung, kidney and plasma and
accumulating drug concentrations in the liver,
spleen and tumor. The majority injected dose
appeared in the liver (56%) and only 1.6% in the
tumour at 48 hrs post injection, confirming that
nanoparticles have a great tendency to be
captured by liver. This indicates the greatest
challenge of using nanoparticles for tumour
targeting is to avoid particle uptake by
mononuclear phagocytic system (MPS) in liver
and spleen.
Such
propensity
of
MPS
for
endocytosis/phagocytosis of nanoparticles
provides an opportunity to effectively deliver
therapeutic agents to these cells. This
biodistribution can be of benefit for the
chemotherapeutic treatment of MPS- rich
organs/tissues localized tumors like hepato-
carcinoma, hepatic metastasis arising from
digestive tract or gynaecological cancers,
brochopulmonary tumors, primitive tumors and
metastasis, small cell tumors, myeloma and
leukemia. It has been proved that using
doxorubicin loaded conventional nanoparticles
was effective against hepatic metastasis model
in mice. It was found there was greater reduction
in the degree of metastasis than when free drug
was used. The underlying mechanism
responsible for the increased therapeutic
efficacy of the formulation was transfer of
doxorubicin from healthy tissue, acting as a drug
reservoir to the malignant tissues 50. Histological
examination
showed
a
considerable
accumulation of nanoparticles in the lysosomal
vesicles of Kupffer cells, whereas nanoparticles
could not be clearly identified in tumoral cells 50.
Thus Kupffer cells, after a massive uptake of
nanoparticles by phagocytosis, were able to
induce the release of doxorubicin, leading to a
gradient of drug concentration, favorable for a
prolonged diffusion of the free and still active
drug towards the neighboring metastatic cells 50.
When conventional nanoparticles are used as
carriers in chemotherapy, some cytotoxicity
against the Kupffer cells can be expected, which
would result in deficiency of Kupffer cells and
naturally lead to reduced liver uptake and
decreased therapeutic effect with intervals of
less than 2 weeks administration 51. Moreover,
conventional nanoparticles can also target bone
marrow (MPS tissue), which is an important but
unfavorable site of action for most anticancer
drugs because chemotherapy with such carriers
may increase myelosuppresive effect. Therefore,
the ability of conventional nanoparticles to
enhance anticancer drugs efficacy is limited to
targeting tumors at the level of MPS-rich organs.
Also,
directing
anticancer
drug-loaded
nanoparticles to other tumoral sites is not
feasible if a rapid clearance of nanoparticles
occurs shortly after intravenous administration.
Long circulating nanoparticles
To be successful as a drug delivery system,
nanoparticles must be able to target tumors
which are localized outside MPS-rich organs. In
the past decade, a great deal of work has been
devoted to developing so-called “stealth”
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Trop J Pharm Res, June 2006; 5 (1)
568
particles or PEGylated nanoparticles, which are
invisible to macrophages or phagocytes 52. A
major breakthrough in the field came when the
use of hydrophilic polymers (such as
polyethylene glycol, poloxamines, poloxamers,
and polysaccharides) to efficiently coat
conventional nanoparticle surface produced an
opposing effect to the uptake by the MPS 52, 53.
These coatings provide a dynamic “cloud” of
hydrophilic and neutral chains at the particle
surface which repel plasma proteins 54 55. As a
result, those coated nanoparticles become
invisible to MPS, therefore, remained in the
circulation for a longer period of time. Hydrophilic
polymers can be introduced at the surface in two
ways, either by adsorption of surfactants or by
use of block or branched copolymers for
production of nanoparticles 51, 52.
Studies show nanoparticles containing a coat of
PEG not only have a prolonged half-life in the
blood compartment but also be able to
selectively extravasate in pathological sites such
as tumors or inflamed regions with a leaky
vasculature 51. As a result, such long-circulating
nanoparticles have increased the potential to
directly target tumors located outside MPS-rich
regions 51. The size of the colloidal carriers as
well as their surface characteristics are the
critical to the biological fate of nanoparticles. A
size less than 100 nm and a hydrophilic surface
are essential in achieving the reduction of
opsonisation reactions and subsequent
clearance by macrophages 52. Coating
conventional nanoparticles with surfactants or
PEG to obtain a long-circulating carrier has now
been used as a standard strategy for drug
targeting
in vivo.
Extensive efforts have been devoted to
achieving “active targeting” of nanoparticles in
order to deliver drugs to the right targets, based
on molecular recognition processes such as
ligand-receptor or antigen-antibody interaction.
Considering that fact that folate receptors are
over expressed on the surface of some human
malignant cells and the cell adhesion molecules
such as selectins and integrins are involved in
metastatic events, nanoparticles bearing specific
ligands such as folate may be used to target
ovarian carcinoma while specific peptides or
carbohydrates may be used to target integrins
and selectins 56. Oyewumi et al demonstrated
that the benefits of folate ligand coating were to
facilitate tumor cell internalization and retention
of Gd-nanoparticles in the tumor tissue 57.
Targeting with small ligands appears more likely
to succeed since they are easier to handle and
manufacture. Furthermore, it could be
advantageous when the active targeting ligands
are used in combination with the long-circulating
nanoparticles to maximize the likelihood of the
success in active targeting of nanoparticles.
Reversion of multidrug resistance in tumour
cells
Anticancer drugs, even if they are located in the
tumour interstitium, can turn out to be of limited
efficacy against numerous solid tumour types,
because cancer cells are able to develop
mechanisms of resistance
58. These
mechanisms allow tumours to evade
chemotherapy. Multidrug resistance (MDR) is
one of the most serious problems in
chemotherapy. MDR occurs mainly due to the
over expression of the plasma membrane p-
glycoprotein (Pgp), which is capable of extruding
various positively charged xenobiotics, including
some anticancer drugs, out of cells 58. In order to
restore the tumoral cells’ sensitivity to anticancer
drugs by circumventing Pgp-mediated MDR,
several strategies including the use of colloidal
carriers have been applied. The rationale
behind the association of drugs with colloidal
carriers, such as nanoparticles, against drug
resistance derives from the fact that Pgp
probably recognizes the drug to be effluxed out
of the tumoral cells only when this drug is
present in the plasma membrane, and not when
it is located in the cytoplasm or lysosomes after
endocytosis 59 60.
Nanoparticles for oral delivery of peptides
and proteins
Significant advances in biotechnology and
biochemistry have led to the discovery of a large
number of bioactive molecules and vaccines
based on peptides and proteins. Development of
suitable carriers remains a challenge due to the
Mohanraj & Chen
Trop J Pharm Res, June 2006; 5 (1)
569
fact that bioavailability of these molecules is
limited by the epithelial barriers of the
gastrointestinal tract and their susceptibility to
gastrointestinal degradation by digestive
enzymes. Polymeric nanoparticles allow
encapsulation of bioactive molecules and protect
them against enzymatic and hydrolytic
degradation. For instance, it has been found that
insulin-loaded nanoparticles have preserved
insulin activity and produced blood glucose
reduction in diabetic rats for up to 14 days
following the oral administration 61.
The surface area of human mucosa extends to
200 times that of skin 62. The gastrointestinal
tract provides a variety of physiological and
morphological barriers against protein or peptide
delivery, e.g., (a) proteolytic enzymes in the gut
lumen like pepsin, trypsin and chymotrypsin; (b)
proteolytic enzymes at the brush border
membrane (endopeptidases); (c) bacterial gut
flora; and (d) mucus layer and epithelial cell
lining itself 63. The histological architecture of the
mucosa is designed to efficiently prevent uptake
of particulate matter from the environment. One
important strategy to overcome the
gastrointestinal barrier is to deliver the drug in a
colloidal carrier system, such as nanoparticles,
which is capable of enhancing the interaction
mechanisms of the drug delivery system and the
epithelia cells in the GI tract. .
Targeting of nanoparticles to epithelial cells
in the GI tract using ligands
Targeting strategies to improve the interaction of
nanoparticles with adsorptive enterocytes and
M-cells of Peyer’s patches in the GI tract can be
classified into those utilizing specific binding to
ligands or receptors and those based on non-
specific adsorptive mechanism. The surface of
enterocytes and M cells display cell-specific
carbohydrates, which may serve as binding sites
to colloidal drug carriers containing appropriate
ligands. Certain glycoproteins and lectins bind
selectively to this type of surface structure by
specific receptor-mediated mechanism. Different
lectins, such as bean lectin and tomato lectin,
have been studied to enhance oral peptide
adsorption 64 65. Vitamin B-12 absorption from
the gut under physiological conditions occurs via
receptor-mediated endocytosis. The ability to
increase oral bioavailability of various peptides
(e.g., granulocyte colony stimulating factor,
erythropoietin) and particles by covalent coupling
to vitamin B-12 has been studied 66, 67. For this
intrinsic process, mucoprotein is required, which
is prepared by the mucus membrane in the
stomach and binds specifically to cobalamin.
The mucoprotein completely reaches the ileum
where resorption is mediated by specific
receptors.
Absorption enhancement using non-specific
interactions
In general, the gastrointestinal absorption of
macromolecules and particulate materials
involves either paracellular route or endocytotic
pathway. The paracellular route of absorption of
nanoparticles utilises less than 1% of mucosal
surface area. Using polymers such as chitosan
68, starch 69 or poly(acrylate) 70 can increase the
paracellular permeability of macromolecules.
Endocytotic pathway for absorption of
nanoparticles is either by receptor-mediated
endocytosis, that is, active targeting, or
adsorptive endocytosis which does not need any
ligands. This process is initiated by an unspecific
physical adsorption of material to the cell surface
by electrostatic forces such as hydrogen bonding
or hydrophobic interactions 71. Adsorptive
endocytosis depends primarily on the size and
surface properties of the material. If the surface
charge of the nanoparticles is positive or
uncharged, it will provide an affinity to adsorptive
enterocytes though hydrophobic, whereas if it is
negatively charged and hydrophilic, it shows
greater affinity to adsorptive enterocytes and M-
cells. This shows that a combination of size,
surface charge and hydrophilicity play a major
role in affinity. This is demonstrated with poly
(styrene) nanoparticles and when it is
carboxylated 72.
Nanoparticles for gene delivery
Polynucleotide vaccines work by delivering
genes encoding relevant antigens to host cells
where they are expressed, producing the
antigenic protein within the vicinity of
professional antigen presenting cells to initiate
immune response. Such vaccines produce both
Mohanraj & Chen
Trop J Pharm Res, June 2006; 5 (1)
570
humoral and cell-mediated immunity because
intracellular production of protein, as opposed to
extracellular deposition, stimulates both arms of
the immune system 73. The key ingredient of
polynucleotide vaccines, DNA, can be produced
cheaply and has much better storage and
handling properties than the ingredients of the
majority of protein-based vaccines. Hence,
polynucleotide vaccines are set to supersede
many conventional vaccines particularly for
immunotherapy. However, there are several
issues related to the delivery of polynucleotides
which limit their application. These issues
include efficient delivery of the polynucleotide to
the target cell population and its localization to
the nucleus of these cells, and ensuring that the
integrity of the polynucleotide is maintained
during delivery to the target site.
Nanoparticles loaded with plasmid DNA could
also serve as an efficient sustained release gene
delivery system due to their rapid escape from
the degradative endo-lysosomal compartment to
the cytoplasmic compartment 74. Hedley et al. 75
reported that following their intracellular uptake
and endolysosomal escape, nanoparticles could
release DNA at a sustained rate resulting in
sustained gene expression. This gene delivery
strategy could be applied to facilitate bone
healing by using PLGA nanoparticles containing
therapeutic genes such as bone morphogenic
protein.
Nanoparticles for drug delivery into the brain
The blood-brain barrier (BBB) is the most
important factor limiting the development of new
drugs for the central nervous system. The BBB
is characterized by relatively impermeable
endothelial cells with tight junctions, enzymatic
activity and active efflux transport systems. It
effectively prevents the passage of water-soluble
molecules from the blood circulation into the
CNS, and can also reduce the brain
concentration of lipid-soluble molecules by the
function of enzymes or efflux pumps 76.
Consequently, the BBB only permits selective
transport of molecules that are essential for brain
function.
Strategies for nanoparticle targeting to the brain
rely on the presence of and nanoparticle
interaction with specific receptor-mediated
transport systems in the BBB. For example
polysorbate 80/LDL, transferrin receptor binding
antibody (such as OX26), lactoferrin, cell-
penetrating peptides and melanotransferrin have
been shown capable of delivery of a self non
transportable drug into the brain via the chimeric
construct that can undergo receptor-mediated
transcytosis 77-81. It has been reported
poly(butylcyanoacrylate) nanoparticles was able
to deliver hexapeptide dalargin, doxorubicin and
other agents into the brain which is significant
because of the great difficulty for drugs to cross
the BBB 77. Despite some reported success with
polysorbate 80 coated NPs, this system does
have many shortcomings including desorption of
polysorbate coating, rapid NP degradation and
toxicity caused by presence of high
concentration of polysorbate 80.37. OX26 MAbs
(anti-transferrin receptor MAbs), the most
studied BBB targeting antibody, have been used
to enhance the BBB penetration of lipsosomes
82. However, recently, Ji
et al. demonstrated that
brain uptake of lactoferrin, an iron-binding
glycoprotein belonging to the transferrin (Tf)
family, is twice that of OX26 and transferrrin
in
vivo 79. It is possible soon we will see these BBB
specific molecules used for targeting
nanoparticles to the brain.
CONCLUSION
The foregoing show that nanoparticulate
systems have great potentials, being able to
convert poorly soluble, poorly absorbed and
labile biologically active substance into
promising deliverable drugs. The core of this
system can enclose a variety of drugs, enzymes,
genes and is characterized by a long circulation
time due to the hydrophilic shell which prevents
recognition by the reticular-endothelial system.
To optimize this drug delivery system, greater
understanding of the different mechanisms of
biological interactions, and particle engineering,
is still required. Further advances are needed in
order to turn the concept of nanoparticle
technology into a realistic practical application as
the next generation of drug delivery system.
Mohanraj & Chen
Trop J Pharm Res, June 2006; 5 (1)
571
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