1. methods include chemical modifications like change of


Particle size reduction for enhanced drug bioavailability

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For any solid dosage form when it administrated orally either
(tablet or capsule), it needs to be dissolve
and then absorbed to the body tissue to exert
its action. was found in some oral dosage form when it administrated orally, the
absorption rate is so fast but the dissolution rate is slow it’s called in this
case dissolution rate limited which mean that the drug to be enter the bloodstream depend on the dissolution rate (Dunne
et al., 1999).

Aqueous solubility could be considered as a major challenge facing
drug formulation, especially in optimizing immediate-released oral dosage forms.
Most of the new drug entities are poorly water soluble, so increasing drug
solubility can greatly affect their dissolution and consequently their oral
absorption as well as the bioavailability. According to Biopharmaceutics
Classification System (BCS), the limiting factors that govern the rate and
extent of oral absorption of a drug are aqueous solubility and permeability
through biological membranes. The aqueous solubility of several drugs have a
greater role in determining their permeability and absorption rate, especially
in the case of class II drugs (high permeability and low solubility) as well as
class IV drugs (low permeability and low solubility) (Dunne
et al., 1999; Chen et al., 2011).

Several approaches were investigated
to enhance aqueous solubility of pharmaceutical active ingredients (APIs), and
its bioavailability. These methods include chemical modifications like change of pH, use of buffer, derivatization,
complexation, solid dispersion, and salt formation
or using physical modifications like particle size reduction(Savjani
et al., 2012; Murtaza, 2012).

It has been shown that the
solubility is strongly related the particle size; as
when the particle size became smaller, the surface area will increase, which
lead to be 
more interact with the
solvent and consequently increases its solubility (Fig.1).So it is clearly seen
that the particles in nanometer size have higher dissolution than the
micronized particles (Savjani
et al., 2012).




Figure 1: Relation between particle size and surface area.


According to the Noyes–Whitney equation, decreasing drug particle
size to nanometer range will increase the surface area, which is expected to
result in increasing the drug dissolution rate (Szunyogh
et al., 2013).




D is the diffusion coefficient of the solute, S the surface area, h
the thickness of the diffusion layer, Cs the solubility of the drug, and C the
concentration of solute or drug in the bulk solution.


1.2.   Concept of nanotechnology

Formulation of drug substance as nanoparticles by different
technique gained a considerable attention as an advanced technology that help
to improve the poorly dissolute drugs (Jia,

In the past 20 years are widely
increase more than before and that lead to increase the pharmaceutical company
that developed product in anonize to  more than 150 companies (Zhang
et al., 2008).

In in 1994, Oncospar® which
a product manufacture by Enzon was the first nanoparticle that received FDA
approval as nanoparticle formulation for acute lymphocytic leukemia treatment (Duncan,
2006). Also
in 1995, Doxil® ( liposome-encapsulated doxorubicin), which manufactured
by OrthoBiotech, was introduced in the marker as the first FDA-approved liposome
used treatment of HIV-related Kaposi’s sarcoma, and then approved for the
treatment of ovarian cancer and multiple myeloma (Barenholz,
2012; Petros
and DeSimone, 2010). In
2005, Abraxane® , which was manufactured by Abraxis/AstraZeneca
was the approved by FDA as the first protein-based nanoparticle used for the
treatment of metastatic breast cancer (Petros
and DeSimone, 2010).

After that many products were
approved for the treatment of cancer, pain, and infectious diseases like
Fenofibrate tablet as Nanocrystals used for hypercholesterolemia and Paclitaxel
Powder for suspension for infusion as nanoparticles used for Breast neoplasma and
many other example that result  to
increase the focus of developing product in nanoparticles (Hafner
et al., 2014).


1.3.   Medicinal  and pharmaceutical applications of

In recent years, applications of nanoparticles are increase in our
life especially the pharmaceutical and medicinal
fields. Pharmaceutical applications include drug delivery, cancer therapy and
gene delivery systems, medicinal application like imaging, sensing (Singh
et al., 2008).

In cancer therapy, nanomaterials can be used in diagnosis and  prevention of different  types of cancer such as bone cancer, colon
cancer, prostate cancer, gastric cancer, leukemia, bladder cancer, colorectal
cancer and it also can used for treatment by using nanoscale biological
markers, immunotherapy, photodynamic therapy, stem cells, and anticancer
nanodrugs (Nalwa
and Webster, 2007). Breast cancer is one of most cause of death in women, so there
are different normal way to diagnosis it like ultrasound and optical but using
novel nanomaterials such as using contrast agents as biological imaging, which
can play a better role in for early breast cancer detection, screening,
diagnosis, and prevention because nanomaterials offer significant contrast for
biological imaging that used for breast and also for other types of cancer (Singh
and Nalwa, 2011).

One of the medicinal application of nanoparticles are the fluorescent
nanoparticles that used for biological detection and imaging application that
uses nanoparticles which help tracking and cell-tissue interactions of stem
cells which labeled with fluorescent nanoparticles using magnetic resonance
technique (Chen,

On the other side, one of the pharmaceutical application of
nanoparticles is using it as drug delivery systems as lipid nanoparticles,
liposomes, genes, polysaccharides, and polymeric nanomicelles which can
delivered by oral, pulmonary, ocular, and dermal routes. Nanoparticles are
10?100 nm in dimension can easily cross the blood-brain barrier (BBB) to treat various
brain disease like cancer, nervous system diseases, and other various types of
cancer (Nalwa,

 One of the most importance advantages
of successful formation is the delivery of drug in certain place in the body
that is needed for treatment and minimize reaching other places which minimizes
drug side effects especially in cancer
treatment where the tumor may be localizer in certain place in various organs
so by using drug delivery lead
to increase the amount of drug that reach the desire organ and on the other
hand the amount of drug that reach the undesired organ will be decrease (De Jong and Borm, 2008).

Doxil® a liposome-encapsulated doxorubicin, that is used
for treatment of HIV-related Kaposi’s sarcoma, has advantages over free
doxorubicin are high efficacy and low cardiotoxicity. These advantages are due to
passive targeting of tumour due to leaky tumour vasculature that decrease the concentrations
of free doxorubicin in healthy tissue sites (Ning
et al., 2007).


Nanonization techniques: 5

Recently, nanonization techniques arise to overcome the problem of
poor aqueous solubility of many new drugs in water by enhancing its dissolution
rates and bioavailability, and it will also help to decrease their systemic
side-effects. These techniques despond on decreasing drug particle size to
nanometer range which lead to increase in the surface area that results in
increasing the drug dissolution rate, or by changing the crystalline forms, or
by preparing nanomaterials that used as carriers for controlled release (Junghanns
and Muller, 2008; Marcato and Duran,

Nanonization could be prepared by two different techniques,
depending on the technology used to achieve the nanonization. One of the
techniques can be prepared by particle size reduction of large crystals which
called “top-down techniques” and this can be done by either high-pressure
homogenization or media milling (Van Eerdenbrugh et al., 2008).

The second nanonization technique depend on increasing the particle
size from small to large size by precipitation of dissolved molecules which is
referred as “bottom-up techniques” (Van Eerdenbrugh et al., 2008).


Top-down nanonization techniques

Top-down techniques are most commonly used for nanonization which
can be applied using either media milling and/or high-pressure homogenization
(HPH) but it often requires a long process time to reduce particle sizes below
100 nm (Sinha
et al., 2013). High-pressure homogenization (HPH) has
been widely used in the pharmaceutical industry since the mid-1990s when
SkyePharma developed Dissocubes® piston-gap homogenizer that is a
high energy process in which reduction of particles size of drug is achieved by
repeatedly cycling to 200 plus cycles, with the aid of a piston, a drug
suspension through a very thin gap at high velocity, around 500 m/s, and
pressure, 1000-1500 bars. (Keck
and Muller, 2006; Loh et al., 2015)

Recently, HPH is used for preparing drug nanocrystals such as
Triglide® (Fenofibrate), which clinically approved for treatment of
hypercholesterolemia or hyperlipidemia .
HPH is used also used for preparing drug like “Nanopure”, “Nanocrystal”, “NanomorphTM”and “Nanoedge” (Keck
and Muller, 2006; Chen et al., 2011)

Nanoedge™ technology which is HPH techniques combined with
precipitation techniques. In this technique, the drug is first dissolved in a
water-miscible alcoholic solvent like methanol or ethanol, then cause it to
precipitate by added water. The particles that precipitated are then
homogenized. The homogenization is used reduce to the size and size
distribution of the precipitated particles that help to minimizing the probability
of crystal growth and improving the stability of the nanosuspension during
storage (Loh
et al., 2015)

Ball milling (also called Nanocrystals or Nanosystems) is another
popular size reduction technique used top-down concept
which was first developed and reported since 1995 by Liversidge co-workers (Liversidge
and Cundy, 1995).

For the production of nanoparticles, it is based on mixing the drug
powder with the dispersion media (mostly water) and a suitable stabilizer that
helps to avoid or minimize the particle aggregation after the preparation of
nanoparticles (Chen
et al., 2011; Salazar et al., 2014; Loh
et al., 2015). The
milling media (balls) are available in many types like zirconium dioxide beads,
silicium nitride beads and polysterene beads. High shear forces of the
balls results in nanonizing will be obtained when vessel jackets rotate at a very
high speed. Several parameters can affect the resulting particle size of ball
milling nanonization methods such as the amount of drug, type, and concentration
of stabilizers, the amount and size of the ball, speed, time, and temperature. Many
drugs have been nanonized using this method for the production of drug
nanoparticles like fenofibrate, naproxen and ibuprofen (Chen
et al., 2011; Loh et al., 2015; Salazar
et al., 2014).

Some of these drugs are available in the pharmaceutical markets as
tablet forms, such as fenofibrate, ibuprofen, and paliperidone palmitate (Loh
et al., 2015).


Bottom up
nanonization techniques

In the other hand of  top-down techniques, bottom-up techniques
which produce nanoparticles from drug molecules in solution by building them
and can be done by controlled precipitation (or crystallisation) (Chan
and Kwok, 2011). Bottom-up techniques have advantages over the top-down techniques
including that they require low energy processes, can  operate using simple instruments, cheaper and
can be operated at a low temperature, that appropriate techniques for
thermolabile drugs (Rasenack
and Muller, 2004)

Usually, precipitation methods are used to prepare the bigger
particle size which is in micro range size but it also can be used to prepare small
particle size in nanosize range with many method .the researchers in the past two
decade tried to use different methods like precipitation using liquid solvent (antisolvent
addition), precipitation in presence of supercritical fluid, precipitation by
removal of solvent and precipitation in presence of high energy processes (Sinha
et al., 2013)

Precipitation by liquid solvent method (antisolvent addition) depends
on dissolving the drug substance in solvent better to be in water miscible
solvent in which the drug has a suitable solubility. Then, the prepared solution
will be mixed. After that, with an antisolvent addition (water usually) by
using mixing forces, which should be miscible with the solvent phase. In
selection of solvent and antisolvent, the order and volume ratio of solvent–antisolvent
mixing are one of the critical process parameters of precipitation (Sinha
et al., 2013; Savjani et al., 2012).

NanoMorph® technology (develop by Abbott GmbH & Co.KG,
Ludwigshafen, Germany) is an example of precipitation by liquid solvent method
which based on preparing a suspension of a drug in organic solvents that form
solution by heating in mixing chamber, then it mixed quickly with a cooled
aqueous solution that contain stabilizer that help to nucleation of the
particles leading to form spherical amorphous particle of nanometer size (Shi
et al., 2009).

Precipitation using supercritical fluids (SCF) are preferred for
commercial use because it is cheap, usually available, neither toxic nor
flammable, that’s why it is often used in particle engineering, and all drugs not
only the thermolabile drugs. The solvent that generally used as supercritical
solvents include carbon dioxide, ammonia, fluoroform, ethane and ethylene. Some
of these solvents may limit their uses in pharmaceutical applications because
of toxicity and flammability. Many different processes to prepare drug nanoparticles
depend on gas anti-solvent recrystallization (GAS), rapid expansion of
supercritical solutions (RESS) and supercritical antisolvent (SAS). That in
general based on supercritical fluid technologies (de
Waard et al., 2011; Sinha et al., 2013).

Adami et al. used Supercritical AntiSolvent precipitation (SAS) to micronized
particles of nalmefene hydrochloride using using EtOH as solvent that result to
produce particles in the range 1–5 ?m (Adami
et al., 2008).

Precipitation by removal of solvent is another method to prepare nanoparticles
by bottom-up technique that is based on the use of either conventional solvent
removal techniques like freeze-drying and spray drying or the use of special
freezing techniques like spray freezing into liquid (SFL) method.(Savjani
et al., 2012). Precipitation coupled with high energy processes (combination
technologies) are one of the process to prepare nanoparticles by bottom-up
techniques which based on use the precipitation method with a high energy
process such as high pressure homogenization HPH like Nanoedge™ technology (Loh
et al., 2015).


Stabilization of nanoparticles

Preparing the drug in nanoparticles often lead to increase the
surface area of the nanoparticles compared to microparticles, which is usually
accompanied by change in the stability of the particles (Patravale
et al., 2004). Physical Instability problems like sedimentation, crystal growth,
agglomeration, or change of crystallinity state are one of the most common
problems with nanonization that should be limited or avoided (Wu
et al., 2011).  Dry state like solid dosage
forms usually have a better stability than suspensions. Therefore, to limit or
avoid the stability problem of nanosuspension, nanosuspension should be change to
powder form (Patravale
et al., 2004; Abdelwahed et al.,

Agglomeration is one of the physical instability phenomena that affect
the potential of nanonization. It might be accompanied with the preparation of
the drug in nanoparticles which leads to an increase in the free energy of the
system resulting in aggregation of the particles to form a large particle (Wang
et al., 2013; Verma et al., 2011).

Crystal growth or Ostwald ripening is also one of physical instability
phenomena that happen because the dependence of particles solubility on their
sizes, where large particles have lower saturation solubility than small
particle size that lead to concentration gradient between large and small
particle which consequence accumulate to form large particle (Wu
et al., 2011). Narrowing particle size distribution can also help to limit the Ostwald
ripening, because when the particle size is uniform, the Ostwald ripening will
be at a minimum rate. Mechanical agitation temperature and also affect Ostwald
ripening (Rabinow,

Change of crystalline state between amorphous and crystalline state
is also one of the problems that lead to instability of the nanoparticles, where
in top down techniques, the particles tend to form partially amorphous while it
form completely amorphous in case of bottom-up techniques (Lindfors
et al., 2007). Nanoparticles
in amorphous form are usually more soluble and have better dissolution rate
than crystalline form, which make them more stable but the nanoparticles in
amorphous form usually thermodynamically unstable and transfer to crystalline
form by aging (Wang
et al., 2013).

Chemical stability problems like hydrolysis and oxidation can
also affect the stability of nanosuspension (Labille
and Brant, 2010). One of The popular methods to improve the chemical stability is either
by changing the nanosuspension from liquid state to solid dosage form which a stability
will be achieved than nanosuspension. Another method to enhance chemical
stability of drug during nanonization is the increase the of concentration of
the nanosuspension (Van Eerdenbrugh et al., 2008).

Preparing the drug in nanoparticles often result in increasing the
surface area of the nanoparticles compared to microparticles, which may be
accompanied by a change in behavior issue that can affect the stability the
particles like sedimentation, crystal growth, agglomeration, or change of
crystallinity state which should be limited or avoided (Patravale
et al., 2004; Wu et al., 2011).

One of the important approach to stabilize the nanoparticles  is the addition of suitable stabilizer to the
formulation during nanonization process (Peltonen
and Hirvonen, 2010).

Addition of a stabilizer to the nanosuspension formulation can play
an important role to limit agglomeration by reducing the free energy of the
system during nanosization process. The stabilizer may be used either (i)
surfactants such as sodium lauryl sulfate and poloxamer 188, or (ii) polymers
such as hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP)(Verma
et al., 2011; Wang et al., 2013).

 The stabilizer that may be
used either, surfactants, polymers or mixture of both but the selection of best
stabilizer is mainly depend the drug used (Liu
et al., 2011).

The first type of stabilizers that popularly used are surfactants, which
can be either nonionic like tween 80 or ionic like sodium lauryl sulfate and
docusate sodium  (DOSS), but the toxicity
the cationic surfactants limit their application in oral preparation (Kesisoglou
et al., 2007).

Ionic surfactants can act as stabilizers by creating of surfaces with
a charge sufficient enough for nanoparticle stabilization, where the existing
of other charged materials can minimize the surface charge of the particles and
decreased electrostatic repulsion which lead to agglomeration (Palla
and Shah, 2000). On the other hand, nonionic stabilizers are most common used but
they do not provide a significant repulsive barrier against agglomeration like
ionic surfactants, but these work as a stabilizer based on steric affect that
acts as physical barrier around the nanoparticles that decrease the contact
between the particles (Palla
and Shah, 2002).

Yang et al use tween 80 as nonionic surfactant with fluticasone and
budesonide to evaluate the efficiency of particle size reduction of nanosuspension
using wet-milling method (used single-sized glass beads with 0.50–0.75 mm
diameters). Nanosuspension for both compounds
exhibited good physical/chemical properties for pulmonary delivery. The pharmacokinetic studies after the administration of
nanosuspensions showed deep lung deposition and fast lung absorption, with
solubility playing an important role in lung retention and duration of action (Yang
et al., 2008)

The other types of stabilizers are polymeric stabilizers like polyvinylpyrrolidone
(PVP), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl cellulose (HPC),
and polyvinyl alcohol (PVA) (Van Eerdenbrugh et al., 2008).

Polymeric stabilizers act like nonionic stabilizers by steric effects,
but with higher adsorption potential than nonionic  surfactant (Palla
and Shah, 2002). Many factors related to stabilizer can affect the optimum
efficacy of the nanoparticles stabilizers .One of the important parameter is the
concentration of stabilizer, where the amount that used to the nanoparticles
preparation should be optimized. For example, in surfactant stabilizers, the
amount that should be used at concentrations below the critical micelle
concentration (CMC), while concentration should be enough to give the steric
repulsion between the nanoparticles in suspension. (Peltonen
and Hirvonen, 2010; Wang et al., 2013).

 Verma et al. evaluated various
stabilizers include (SLS, PVP K-30, Pluronic F-68 and F-127, Tween 80 and
different (HPMCs)) to study their effects on the
formation and stability of ibuprofen nanosuspension using  different top-down and bottom-up approaches.
Thy find higher particle sizes in the case of suspensions made with SLS, Tween
80 and Pluronic F-127. The higher increase in particle size suggests that
Ostwald ripening may be a key driving force .(Verma
et al., 2009)

of nanonization is also critical parameter that should be controlled during
nanoparticles preparation.