Advanced Drug Delivery Reviews 47 (2001) 3–19

www.elsevier.com / locate / drugdeliv

Nanosuspensions as particulate drug formulations in therapy

Rationale for development and what we can expect for the
future
¨ *, C. Jacobs, O. Kayser
R.H. Muller
¨ Berlin, Institut f ur
Freie Universitat ¨ Pharmazie, Pharmazeutische Technologie, Biopharmazie and Biotechnologie, Kelchstraße 31,
D-12169 Berlin, Germany

Abstract

An increasing number of newly developed drugs are poorly soluble; in many cases drugs are poorly soluble in both
aqueous and organic media excluding the traditional approaches of overcoming such solubility factors and resulting in
bioavailability problems. An alternative and promising approach is the production of drug nanoparticles (i.e. nanosuspen-
sions) to overcome these problems. The major advantages of this technology are its general applicability to most drugs and
its simplicity. In this article, the production of nanoparticles on a laboratory scale is presented, special features such as
increased saturation solubility and dissolution velocity are discussed, and special applications are highlighted, for example,
mucoadhesive nanosuspensions for oral delivery and surface-modified drug nanoparticles for site-specific delivery to the
brain. The possibilities of large scale production — the prerequisite for the introduction of a delivery system to the market —
are also discussed.  2001 Elsevier Science B.V. All rights reserved.

Keywords: Nanosuspension; Homogenisation; Drug targeting; Drug delivery; Parasitic infection; Leishmania; Poor solubility

Contents

1. Introduction ............................................................................................................................................................................ 4
2. Production of nanosuspensions on a laboratory scale.................................................................................................................. 4
3. Physical and chemical properties of nanosuspensions ................................................................................................................. 6
3.1. Characterization of nanosuspensions.................................................................................................................................. 6
3.2. Physical and chemical properties ....................................................................................................................................... 8
4. Surface modification of nanosuspensions .................................................................................................................................. 9
5. Biological properties of nanosuspensions and medical indications............................................................................................... 11
5.1. Oral administration of nanosuspensions ............................................................................................................................. 11
5.2. Parenteral nanosuspensions — intravenous administration route .......................................................................................... 12
6. Large-scale production of nanosuspensions ............................................................................................................................... 15
7. Perspectives ............................................................................................................................................................................ 17
References .................................................................................................................................................................................. 17

*Corresponding author. Tel.: 149-30-838-50416; fax: 149-30-838-50478.

¨
E-mail address: mpharma@zedat.fu-berlin.de (R.H. Muller).

0169-409X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0169-409X( 00 )00118-6
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
1. Introduction
Poorly soluble drugs are a general problem in
pharmaceutical drug formulation [1]. Typical prob-
lems associated with poorly soluble drugs are a too
low bioavailability and / or erratic absorption. In case
of a too low bioavailability after oral administration,
parenteral administration cannot solve this problem
in many cases. Due to the poor solubility, intraven-
ous injection as a solution is not possible. Parenteral
administration as a micronized product (e.g. i.m. or
i.p.) does not lead necessarily to sufficiently high
drug levels because the solute volume at the injection
site is too low. Low saturation solubility, generally
combined with a low dissolution velocity is the
obstacle preventing sufficiently high blood levels.
Possible exceptions are only drugs being poorly
soluble but highly potent.
Attempts to increase the saturation solubility, and
thus solving the problem, are solubility enhancement
by using solubilization (e.g. mixed micelles as in
Valium MM  for i.v. injection), non-specific or
specific complexation (e.g. addition of poly-
ethylenglycol (PEG) or use of cyclodextrins) and
solvent mixtures (e.g. ethanol–water, up to 20%
ethanol are possible). The limited success of these
attempts is documented by the low number of
products on the pharmaceutical market based on
these principles. The problems to find a suitable
formulation are even greater in case the drugs are
poorly soluble in aqueous media and at the same
time in organic media. This excludes the use of
solvent mixtures a priori.
A general approach used for many years is the
micronization of poorly soluble drugs by colloid
mills or jet mills. The overall particle size dis-
tribution ranges from 0.1 mm to approximately 25
mm, only negligible amount being below 1 mm in the
nanometer range [2]. Micronization increases the
dissolution velocity of the drug due to the increase in
surface area but does not change the saturation
solubility. At very low saturation solubility, the
achieved increase in dissolution velocity does not
lead to a sufficiently high bioavailability.
The next development step was transformation of
the micronized drug powder (i.e. drug microparti-
cles) to drug nanoparticles [3,4]. In the eighties, drug
nanoparticles were produced by Sucker and co-work-
ers [5–7] using a precipitation technique. Precipi-
tation was performed by dissolving the drug in a
solvent and adding this solvent to a non-solvent
(so-called ‘via humida paratum’). The basic chal-
lenge of this technique is that during the precipitation
procedure the growing of the drug crystals needs to
be limited by surfactant addition to avoid formation
of microparticles. The requisites limiting the ap-
plicability of the precipitation technique are the
needs of the drug (a) to be soluble at least in one
solvent and (b) that this solvent needs to be miscible
with a non-solvent. These prerequisites exclude the
processing of drugs which are simultaneously poorly
soluble in aqueous and in non-aqueous media.
In the first generation applying disintegration
techniques, this was achieved by pearl milling lead-
ing to the product NanoCrystals  . This patent-
protected technology was developed in 1990 by
Liversidge et al. [4] and formerly owned by the
company NanoSystems, recently acquired by Elan.
To produce NanoCrystals  , the drug powder is
dispersed in a surfactant solution and the obtained
suspension undergoes a pearl milling process for
hours up to several days.
The second generation products are drug
nanoparticles produced by high pressure homogeni-
zation leading to the so called nanosuspensions
(DissoCubes  ). The nanosuspensions were de-
¨
veloped by Muller et al. [3] in 1994 and the patent
rights owned by DDS (Drug Delivery Services)
GmbH in Germany, now owned by SkyePharma
PLC. This chapter presents the production of
nanosuspensions on a laboratory scale but also on
large industrial scale, discusses their special features,
reviews present applications including bioavailabilty
aspects, especially site-specific delivery and high-
lights the future perspectives. Especially, the po-
tential for targeting to the mucosa of the gastroin-
testinal tract (GIT) after oral administration, and to
the cells of the mononuclear phagocytic system
(MPS) to treat MPS infections are discussed.
2. Production of nanosuspensions on a
laboratory scale
The disintegration principle for obtaining nanosus-
pensions are the cavitation forces created in high
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
pressure homogenizers, e.g. piston-gap homogenizers
like APV Gaulin types (Fig. 1). The drug powder is
dispersed in an aqueous surfactant solution by high
speed stirring. The obtained ‘macro’-suspension is
passed through a high pressure homogenizer apply-
ing typically 1500 bar and three to ten up to a
maximum of 20 passes ( 5 homogenisation cycles).
Generally it is recommended to start with a powder
as fine as possible, that means a jet milled product.
The suspension passes a very small homogenization
gap in the homogenizer, typically having a width of,
for example, approximately 25 mm at 1500 bar (Fig.
2). Due to the narrowness of the gap the streaming
velocity of the suspension increases tremendously,
that means the dynamic fluid pressure increases.
Simultaneously the static pressure on the fluid de-
creases below the boiling point of water at room
¨
Fig. 1. APV LAB 40 (APV Deutschland GmbH, Lubeck, Ger-
many).
5
temperature [8]. In consequence, water starts boiling
at room temperature due to the high pressure, gas
bubbles are formed which implode ( 5 cavitation)
when the fluid leaves the homogenisation gap. These
cavitation forces are strong enough to break the drug
microparticles to drug nanoparticles.
The mean particle size in the nanometer range
obtained by this procedure depends on the pressure
and number of cycles applied, in addition it is
affected by the hardness of the drug itself. For
example, for paclitaxel nanosuspension a mean di-
ameter of 330 nm was reported [9], for clofazemine
600 nm [10,11], and for the drug RMKP22 540 nm
[3,11]. Of course, these diameters depend also on the
production conditions chosen. For example, for
budesonide a pressure of 1500 bar and ten cycles
lead to a mean PCS diameter of 511 nm, increasing
the cycle numbers to 15 reduces the size to 462 nm,
and increasing the pressure to 2500 bar and ten
cycles leads to particles with a diameter of 363 nm
[12]. In summary, a certain size can be produced in a
controlled way by adjusting the production parame-
ters pressure and cycle number accordingly. For each
drug, a minimum size exists which can be achieved
by applying a certain pressure, this minimum size
depends on the power density in the homogenizer
and the hardness of the drug itself.
Homogenizers to be used on a laboratory scale
are, for example, the APV Micron LAB 40 (APV
Deutschland GmbH, Lubeck, ¨ Germany) but also
other piston-gap homogenizers from Avestin (Aves-
tin Inc., Ottawa, Canada) and Stansted (Stansted
Fluid Power Ltd., Stansted, UK). The LAB 40 has a
minimum batch volume of 20 ml and a maximum of
40 ml, thus allowing the cost-effective processing of
even expensive drug materials. Even smaller vol-
umes can be prepared by using, for example, the
Avestin EmulsiFlex-B3 (volume 3.5 ml). The op-
timum particle size of nanosuspension depends on
the nature of the drug (therapeutic field) and the
desired biopharmaceutical properties. In cases where
a very fast dissolution is required, a size of approxi-
mately 100–200 nm is preferred. Where prolonged
dissolution is desired (e.g. mucoadhesive nanosus-
pension for treatment of Cryptosporidium infec-
tions), the mean particle diameter might be placed in
the upper nanometer range, e.g. 800–1000 nm. For
intravenous administration, larger nanosuspensions
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19

Fig. 2. Production of nanosuspensions by high pressure homogenization (I: implosion area, II: boiling area).

are preferred for targeting to the MPS cells (to avoid 3. Physical and chemical properties of
complete dissolution of the particles before they nanosuspensions
reach the macrophages). For a fast dissolution of the
drug in the blood, small nanosuspensions are more 3.1. Characterization of nanosuspensions
suited. It should be pointed out, that a relationship
such as ‘the smaller the drug nanoparticles, the better Essential characterization parameters for nanosus-
the product’ does not exist. The size needs to be pensions are:
tailor-made specific for each therapeutic purpose.
Typical concentrations of solid in the suspensions (1) Size and size distribution
processed are 10 or 20% with Stanstead homogeniz- (2) Particle charge (zeta potential)
ers; however 40% suspensions were also processed (3) Crystalline status
[13]. The solid concentration chosen depends on the (4) Dissolution velocity and saturation solubility.
final application of the nanosuspension. For intraven-
ous injection 10% or even less are sufficient for For surface-modified nanosuspensions a number of
further processing of the nanosuspension to a dry additional parameters have to be investigated to
product (e.g. by granulation to tablets); a higher solid obtain a complete picture, especially with relevance
content is desirable to remove less water. for the in vivo behavior:
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
(5) Adhesion properties (in case of mucoadhesive
particles)
(6) Surface hydrophilicity / hydrophobicity
(7) Interaction with body proteins.
The mean size and width of distribution (polydis-
persity index) is typically determined by photon
correlation spectroscopy (PCS) [14]. The measuring
range of PCS is limited to approximately 3 nm–3
mm. Therefore additionally laser diffractometry (LD)
is used to detect any content of particles in the
micrometer range or aggregates of drug nanoparti-
cles. Depending on the type of equipment employed,
the measuring range is approximately 0.05–80 mm
up to maximum of 2000 mm. The LD data are
volume data, typical characterization parameters are
the diameters 50, 90, 95, and 99% (i.e. a diameter of
99% means that 99% of the volume of the particles
is below the given size in micrometers).
For nanosuspensions to be administered intraven-
ously, an additional analysis by Coulter counter
technique is essential. In contrast to the LD pro-
viding only a relative size distribution, the Coulter
counter gives absolute data (i.e. absolute number of
particles per volume unit for the different size
classes). For intravenous injection the content of
particles larger 5 mm is critical and has to be under a
certain limit. Bearing in mind that the smallest size
of blood capillaries is 5–6 mm, too high for a
number of such particles that would lead to capillary
blockade and embolism. Therefore, this factor needs
to be controlled by Coulter counter analysis.
The crystalline structure of the nanosuspensions
can be assessed by differential scanning calorimetry
(DSC) [12]. This is especially important when a drug
exists in different polymorphic forms. In addition,
the high pressure homogenization technique for
nanosuspensions can be used to particles with an
amorphous fraction, thus leading to an enhancement
of the saturation solubility. The extend of such a
transform can be quantified and its stability during
storage monitored by DSC — preferentially in
combination with X-ray analysis. In general, wide
angle X-ray analysis (WAXS) is employed because it
is easily accessible (in contrast to SAXS).
Particle charge is a stability determining parameter
in aqueous nanosuspensions. It is measured by
7
electrophoresis and typically expressed as electro-
phoretic mobility [(mm / S) /(V/ cm)] or converted to
the zeta potential (mV). For a physically stable
nanosuspension solely stabilized by electrostatic
repulsion, a zeta potential of 630 mV is required as
a minimum [15,16]. In the case of a combined
electrostatic and steric stabilization, as a rough guide
line of 20 mV is sufficient.
Determination of dissolution velocity is important
to assess the benefits compared to the traditional
drug formulation (e.g. coarse powder, micronized
product). To determine the dissolution velocity, the
methods described in the Pharmacopoeia can be used
(e.g. for determination of the saturation solubility
shaking experiments at different temperatures (4, 20,
408C) need to be performed until equilibrium has
been reached). Special feature of the nanosuspen-
sions is an increased saturation solubility. Increased
dissolution velocity and increased saturation solu-
bility — apart from the adhesive properties of
nanosuspensions — are the basic benefit of nanosus-
pensions compared to traditional dosage forms. To a
large extent, these two parameters allow estimation
of the change in in vivo performance (blood profiles,
plasma peaks, bioavailability).
For intravenously injected nanosuspensions, addi-
tional parameters need to be determined which affect
the in vivo fate of the drug nanoparticles. Surface
hydrophilicity / hydrophobicity is considered as one
of the important parameters affecting the in vivo
organ distribution after i.v. injection. The surface
hydrophobicity determines the interaction with cells
prior to phagocytosis [17,18]; in addition, it is a
relevant parameter for the adsorption of plasma
proteins — the key factor for organ distribution
[19,21–23]. To avoid artefacts, the surface hydro-
phobicity needs to be determined in the original
environment of the drug nanoparticles, that means in
aqueous dispersion medium. A suitable technique is
hydrophobic interaction chromatography (HIC), pre-
viously employed to determine the surface hydro-
phobicity of bacteria [24], and then transferred to the
characterization of nanoparticulate drug carriers. For
details, we refer to Refs. [18,25,26].
The qualitative and quantitative composition of the
protein absorption pattern, observed after i.v. in-
jection of the particles, is today generally recognized
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
as the essential key factor for organ distribution
[20–22,27,28]. The protein analysis by 2-D PAGE
was modified and especially adapted to the analysis
of protein adsorption patterns on nanoparticles [20].
Time-dependent adsorption patterns of proteins on
nanoparticles were determined after incubation of
polymeric particles with plasma or serum [20,29,30],
but also after collection of i.v. injected particles in
animals [31,32].
Basic correlations could be established between
the protein adsorption patterns and the organ dis-
tribution, here reflected by MPS cell uptake, certain
particles circulating in the blood [29], particles taken
up by the bone marrow, and particles delivering
drugs to the brain [30,33]. These established correla-
tions can now be fully exploited to produce in a
controlled way target-specific nanosuspensions. For
detailed information about this concept of ‘differen-
tial protein adsorption’, we refer to the relevant
literature and patents [19–22,26,27,32]. In this chap-
ter we only discuss targeting to the MPS cells.
The uptake of i.v. injected nanoparticles by MPS
cells is a natural phenomenon. The particles are
recognized as being foreign and phagocytosed by the
macrophages, mainly in the liver (60–90%), the
spleen (approximately 1–5%) and to a very small
extend by the lung macrophages [18]. Because it is a
natural process, this targeting has been called ‘natu-
ral targeting’ in the literature. Because it happens
automatically, this ‘natural targeting’ is considered to
be achievable very easily. Despite this ‘easiness’, no
commercial products are on the market exploiting
this effect. In addition, it has to be realized that
uptake of the drug nanoparticles has to be extremely
fast to avoid dissolution of the particles. In addition,
it should be as complete as possible, that means
preferably 90–100%. Depending on the nature of the
surfactants used, sometimes it takes 15–30 min [18].
This might be too long for many nanosuspensions of
drugs. Therefore, the surface properties of the
nanosuspensions need to be adjusted this way, so
that fast recognition and uptake takes place. This
means the natural uptake by the MPS cells needs to
be further accelerated and enhanced. The surface
properties need to be changed in a controlled way
such that preferentially adsorbed proteins can medi-
ate the uptake process by macrophages. This can be
done by using HIC in combination with 2-D PAGE.
3.2. Physical and chemical properties
An outstanding feature of nanosuspensions is the
increase in saturation solubility and consequently an
increase in the dissolution velocity of the compound.
This increase in the dissolution velocity takes place
in addition to the increase caused by the enlargement
of the surface area, e.g. exploited in micronized
products. In general, in textbooks, the saturation
solubility is defined as being a compound-specific
constant depending only on the temperature. In case
of polymorphism, the saturation solubility depends
also on the crystalline structure (i.e. the inner
energy) — this means solubility is best for the
polymorphic modification that is characterised by
highest energy and lowest melting point. However
— apart from temperature and crystalline / amor-
phous status — the saturation solubility is also a
function of the particle size. This size-dependency
comes only into effect for particles having a size
below approximately 1 mm — a phenomenon ob-
served in tabletting which leads to an increase of the
intrinsic dissolution rate of such fine drug particles
[34,35]
The increase in saturation solubility can be ex-
plained by the Kelvin and the Ostwald–Freundlich
equation [35]. The Kelvin equation describes the
vapor pressure over a curved surface of a liquid
droplet in gas. The vapor pressure increases with
increasing curvature, which means decreasing par-
ticle size. This equation, describing the transition of
molecules from a liquid phase (droplet) to a gas
phase, can also be applied to the transition of
molecules from a solid phase (drug particle) to a
liquid phase. The vapor pressure is then replaced by
the dissolution pressure. The equilibrium between
dissolving molecules and molecules recrystallizing
on particle surfaces (determining the extend of
saturation solubility) is shifted in favour of the
dissolution process. As a result of this increased
dissolution tendency, the saturation solubility in-
creases. The dependency of the saturation solubility
is also described in the Ostwald–Freundlich equation
comparing the solubilities of two particles with
different diameters [35].
The Noyes–Whitney equation describes the disso-
lution velocity dc / dt which depends on the surface
area A and difference (cs 2 cx) /h (cx — equilibrium
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
concentration in bulk phase, h — diffusional dis-
tance). According to the Prandtl equation, the diffu-
sional distance h is reduced for small particles [35].
The simultaneous increase in saturation solubility cs
and decrease in h leads to an increased concentration
gradient (cs 2 cx) /h, thus enhancing the dissolution
velocity in addition to the surface effect.
A third pronounced property is the adhesiveness
generally described for nanoparticles [36]. There is a
distinct increase in adhesiveness of ultra fine pow-
ders compared to coarse powders. A nice example
from daily life is differently sized sugar. Coarse
sugar crystals adhere less effectively to a cake or
cookie compared to iced sugar. This adhesiveness of
small drug nanoparticles can be exploited for im-
proved oral delivery of poorly soluble drugs. The
oral administration of drugs in form of drug
nanoparticles (NanoCrystals  ) has been reported to
have a full range of positive effects [37]:
(1) Improving bioavailability
(2) Improving dose proportionality
(3) Reducing fed / fasted variability
(4) Reducing inter-subject variability
(5) Enhancing absorbtion rate (both human and
animal data)
These data reported by the company NanoSystems
have been acquired in vivo in animals but also in
humans. Reported increases in bioavailability are
quite impressive, e.g. for danazole nanosuspension
being 82% compared to only 5% for the commercial
danazole macrosuspension [37,38].
An additional feature of nanosuspensions is a
potential change in the crystalline structure, which
means increasing the amorphous fraction in the
particle or even creating completely amorphous
particles. It has been described for tabletting where a
sufficiently high punching pressure can transform a
polymorphic compound to a higher energy modi-
fication. The application of high pressures during the
production of nanosuspensions was found to promote
the amorphous state, e.g. the drug RMKP22 was
found amorphous in the nanosuspension [39]. For
NanoCrystals  prepared by a low energy milling
process, a completely crystalline structure was re-
ported [4,40,41]. The extend of the amorphous
fraction produced in the high pressure homogeniza-
9
tion process depends on the production parameters
(homogenization pressure, number of cycles) but is
also affected by the hardness of the drug. Of course,
the generated structure of the particles needs to be
preserved in case it is not a priori stable, e.g. by
lyophilisation or spray-drying. The increase in the
amorphous fraction leads to a further increase of the
saturation solubility. It is generally known that
amorphous drugs have a higher saturation solubility
then the crystalline form.
Another special feature of nanosuspensions is the
absence of Ostwald ripening [42], meaning physical
long-term stability as an aqueous suspension [11,43].
Ostwald ripening has been described for highly
dispersed systems, which means a reduction in size
of the finest particle fraction and their final dis-
appearance combined with simultaneously growth of
the larger particles. Reasons for the Ostwald ripening
are the different saturation solubilities in the vicinity
of differently sized particles and the concentration
gradient existing between them. Molecules from the
higher concentrated solution around very small par-
ticles diffuse to the vicinity of larger particles where
a lower concentration is present. This leads to
supersaturation and drug crystallization, which
means growth of the larger particles. Simultaneously
the vicinity of smaller particles will be below the
saturation concentration, thus the new drug will be
dissolved — the fine particles are getting smaller.
This is a continuous process finally leading to the
disappearance of the fine particles. The lack of
Ostwald ripening in nanosuspensions is attributed to
the uniform particle size created by the homogeniza-
tion process. The differences in saturation solubility
in combination with the a priori low solubility of the
poorly soluble drug keeps the concentration differ-
ences sufficiently low to avoid the ripening effect.
4. Surface modification of nanosuspensions
The in vivo performance of nanosuspensions can
be improved further above the effects described in
the previous chapter by controlled surface modi-
fication of the drug nanoparticles. The adhesiveness
of the drug nanoparticles is considered to be a major
factor increasing the bioavailability and reducing
variability of absorption and erratic absorption.
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19

Therefore, the approach was taken to further improve
the bioavailability performance by modifying the
particle surface using mucoadhesive polymers.
In the case of intravenous administration for
targeting the drug nanoparticles to the MPS, clear-
ance by the macrophages could be further enhance
above the natural clearance effect by modifying the
particle surface with surfactants or polymers thereby
increasing the adsorption of blood proteins which
promote recognition by the MPS.
To create a certain surface property, the drug
nanoparticles can be produced using special surfac-
tants or polymers as stabilizers in the production
process. Where this is not possible (e.g. lack of
sufficient stabilization effect), the surface modifiers
can be admixed to the nanosuspension. According to
their affinity to the particle surface, they will adsorb
and lead to a modification. The degree of modi-
fication can be followed by physical measurements,
e.g. charge (zeta potential) measurements or hydro-
phobic interaction chromatography (HIC) analysis.
To achieve the desired effect, the surface must not be
completely covered by the surface modifier. To
judge if the adsorbed amount is sufficient, the newly
created surface needs to be characterized in their
relevant parameters for the modification purpose, e.g.
surface hydrophobicity to enhance adsorption of
MPS uptake-promoting proteins (e.g. by HIC and
2-D PAGE) (Fig. 3).
To increase the adhesion to the gut wall of orally
administered nanosuspensions, the particles were
surface modified with polymers, like chitosan and
carbopol. The particles were directly produced using
high pressure homogenization, afterwards the
nanosuspension was incorporated into the polymer
solution.
The surface-properties of i.v. injected particles
determine protein adsorption pattern and organ dis-
tribution resulting from the physical properties of the
used surfactants. There is a correlation surface
hydrophobicity–protein adsorption pattern in the
blood–organ distribution [26,27]. Nanosuspensions

Fig. 3. Cuboid nanosuspension particle coated with stabilizer, e.g. surfactant or polymer. The protein adsorption is a function of the
properties of the stabilizer layer (e.g. charge, hydrophobicity). Depending on the composition of the protein adsorption pattern, MPS uptake
can be enhanced or reduced.
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R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
for i.v. administration were produced using Tween
80  and poloxamer 188 as stabilizer [44]. Com-
position of the nanosuspension was tarazepide 1.0%,
poloxamer 188 1.0% and Tween 80  0.5%, while
ten homogenization cycles with 1500 bar were
applied. The mean particle diameter was 400 nm, the
LD 100% was 3.5 mm.
5. Biological properties of nanosuspensions and
medical indications
5.1. Oral administration of nanosuspensions
First choice of application is oral administration.
When a drug is given orally, the bioavailability and
finally its efficacy depends on the solubility and
absorption in the gastrointestinal tract. In vitro,
highly active compounds have failed in the past
because their poor solubility has limited in vivo
absorption and did not lead to effective therapeutic
concentrations. Simple examples reflecting this prob-
Fig. 4. Transmission electron micrographs of a atovaquone
nanosuspension (medium size: 468 nm).
11
lem of poor solubility combined with low absorption
are the experimental compounds, bupravaquone and
the recently approved drug, atovaquone
(Wellvone  ), used for the treatment of opportunistic
Pneumocystis carinii infections in HIV patients.
Besides this indication, both drugs are also very
effective for the treatment of leishmaniasis, but
require an extremely high dose. Furthermore, ato-
vaquone was recently approved in combination with
proguanil (Malarone  ) for therapy of non-compli-
cated P. falciparum malaria [45].
Atovaquone is given orally three times 750 mg
daily, reflecting a high dose as an antibiotic. The
main reason for the high dose is the low absorption
— only 10–15% of this poorly soluble drug. A way
to improve bioavailability of atovaquone or bup-
ravaquone would be to increase the absorption rate
by formulating as a nanosuspension. Oral administra-
tion of nanosuspensions can overcome this problem
because of the high adhesiveness of drug particles
sticking on biological surfaces — here the epithelial
gut wall — and prolonging the absorption time.
When formulating atovaquone as a nanosuspension
(Fig. 4) and given orally to Leishmania-infected
mice, the parameter for increased absorption is
related to the infectivity score of each animal or it is
related to the reduction of the parasite load in the
liver. In comparison to Wellvone  -treated mice,
containing a micronized drug, nanosuspensions of
atovaquone at equivalent doses reduced infectivity
from 40 to 15% at a reduced concentration of only
7.5 mg / kg [46]. These results reflect the potency of
this technique, reducing drug load from 22.5 mg / kg
(Wellvone  ) to 7.5 mg / kg, but increasing activity
2.5-fold at the same time. Another example for the
effectiveness of nanosuspensions for improving bio-
availability is the application of amphotericin B.
When amphotericin B is given orally in approved
drugs for the treatment of GIT mycosis, the drug is
not or only to a minor extent absorbed. Formulating
this antimycotic and leishmanicidal drug as a
nanosuspension and administering it orally, the num-
ber of L. donovani parasites in vivo was reduced
significantly, indicating a high uptake of the drug in
the gastrointestinal tract. When given amphotericin B
nanosuspensions orally, the infectivity score (IS) was
determined at 25% (control5100% parasite growth).
In comparison to i.v. (!) administered liposomes
12 ¨
R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
entrapping amphothericin B, like Ambisome  (IS5
40%) and commercial crude amphotericin B (mi-
cronized drug particles) with no leishmanicidal ef-
fect, these results confirm the potency for nanosus-
pensions to increase bioavailability. From the back-
ground of finding an oral formulation for Leish-
mania-infected patients, nanosuspensions demon-
strate a new pathway for future therapy.
The adhesiveness of nanosuspensions can not only
be used to improve bioavailability, it is also a smart
way for drug targeting of parasites persisting in the
gastrointestinal tract. Cryptosporidium parvum has
attracted renewed interest, since it has been identified
as the main pathogen causing severe diarrhoea in
immunosuppressant HIV patients. In the US and
Europe, approximately 10–15% of AIDS patients are
suffering from this opportunistic pathogen, and today
there is still no effective therapy or clinically useful
drug. Formulating drugs to combat C. parvum must
cover two main aspects. First, targeting the drug to
the pathogen which is located in the epithelial
membranes of the gut wall, and secondly, increasing
the time for the drug in the GIT to prolong the
pharmacological window with regard to the fast
washing-out during diarrhoea. Bupravaquone
nanosuspensions provide some advantages, and in
TCR-alpha-deficient mice infected with C. parvum
oocysts, the significance of the nanosuspensions is
documented. In comparison to micronized drug
powder, the infectivity score was reduced from 2.0
(negative control group, untreated) to 1.47 for mi-
cronized bupravaquone and even to 1.02 for equiva-
lent nanosuspensions [47]. With respect to the
pathophysiological situation in the infected GIT,
nanosuspensions were incorporated in a mucoadhe-
sive chitosan matrix (0.5%) and the effectiveness
was retested. Infectivity scores of 0.68 in the cecum
and 0.49 in the ileum documented the improved
action of the mucoadhesive nanosuspension delivery
system [48]. Formulating poorly soluble drugs for
the treatment of Cryptosporidium infections or other
GIT infections, like helminths, will be an important
strategy in the future.
In summary, oral administration of nanosuspen-
sions is a drug delivery strategy, not only to improve
bioavailability, but also to target gastrointestinal
bacterial and parasitic infections. We did not discuss
other fields of interest here, but the nanosuspension
technology is considered as a suitable new colon
delivery systems for the treatment of colon cancer,
helminth infections, gastrointestinal inflammation or
GIT associated diseases like sprue (zoeliaki).
5.2. Parenteral nanosuspensions — intravenous
administration route
Infections like tuberculosis, listeriosis, leish-
maniasis, and toxoplasmosis are caused by parasites
residing the macrophages of the MPS, thus being
relatively easily accessible by i.v. injected particles.
The i.v. injected particles are heavily and quickly
taken up by the MPS cells in case they absorb uptake
promoting proteins like apolipoproteins. Fig. 5 shows
a protein adsorption pattern of hydrophobic poly-
styrene particles which are being cleared in vivo very
fast by liver and spleen macrophages.
However, some parasite do also reside in the brain
(CNS). Especially, in trypanosomiasis and toxoplas-
mosis, the brain-localized parasite mostly leads to
relapsing infections if not cured. Therefore, it would
be of importance to target drug nanoparticles via
surface modification to the brain.
Kreuter et al. [49] reported successful targeting of
the peptide, dalargin, to the brain using Tween 80 
surface modified polyisobutylcyanoacrylates
nanoparticles [49]. The mechanism was identified as
being ApoE-mediated and was confirmed for the
nanosuspensions discussed here. Fig. 5 shows a 2-D
PAGE gel of particles having an affinity to the brain
and transporting the drug. The Tween surface-modi-
fied nanoparticles where analyzed by 2-D PAGE and
showed also the characteristic ApoE spot, that is
clearly visible (arrow). At present, surface-modified
melarsenoxide particles (Tween 80  coated) are
under investigation in vivo to treat murine
trypanosomiasis phase II, also called sleeping sick-
ness. The clinically used drug, melarsoprol
(Arsobal  ), has only 3–5% of the administered dose
detectable in the brain fluid. An improved brain
delivery formulation would allow to reduce the
therapeutic dose and toxic side-effects in parallel.
These results would also have an impact on other
CNS-associated diseases, like trypanosomiasis, but
also toxoplasmosis and cerebral malaria.
Administration of drugs via the parenteral route is
critical, i.e. usually accompanied by physical and
 ¨
R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19 13

Fig. 5. (a) 2-D PAGE gel of Tween 80 modified polyisobutylcyanoacrylate nanoparticles (right) versus unmodified nanoparticles (left)
¨ [30]). Tween 80 coated PBCA particles showing preferentially adsorption of ApoE (arrow). (b) Semi-quantitative 2-D
(modified after Luck
¨ [30]).
PAGE analysis of polybutylcyanoacrylate nanoparticles after incubation in citrate plasma (gels from (a), modified after Luck

biological problems, like production under aseptic
conditions, a sophisticated protocol for safety issues,
and, last but not least, biological problems, such as
endotoxins, allergic reactions, and inconvenience for
the patient.
On the other hand, parental administration can not
be avoided in every cases. Sometimes it is favoured
to target drugs to the desired site of action, even
when they show physical problems like poor solu-
bility. Paclitaxel reflects such a problem [6]. Because
of low solubility, it has been formulated for i.v. use
with Cremophor EL. The main disadvantage is the
high risk of allergic reaction during infusion, which
has been often reported in the past. However,
14 ¨
R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
parenteral administration also has its advantages. A
simple but effective drug targeting principle is to
deliver the drug to infected macrophages. A whole
range of human pathogenic microorganisms persist
intracellularly in macrophages, like Mycobacterium
tuberculosis, Listeria monogyna, Leishmania sp.
Application of drugs as particles will exploit the
natural endocytotic mechanism of these mononuclear
cells of grabbing all particular objects, mostly cell
fragments, macromolecular proteins, or microorga-
nisms invading the body. Colloidal drug carriers
systems have attracted interest for many years in
their ability to incorporate different drugs in mi-
croparticles, liposomes, polymer particles, mi-
croemulsions thereby exploiting this natural uptake
mechanism (Fig. 6). Even for the treatment of
Leishmania infections these above-mentioned deliv-
ery systems have been investigate intensively. How-
ever, even today no formulation technique has been
approved and launched to the market. For liposomes,
the main problems are high costs in production or the
absence of a cost-effective and simple scaling-up
technology (see Section 6). Nanosuspensions may
contribute to solve these problems.
Aphidicolin is a fungal metabolite from Nigros-
Fig. 6. Uptake of drugs and intracellular trafficking: free drugs (h) versus nanosuspensions (j). (I) Diffusion, (II) endocytosis, (IIa)
releases of nanoparticles and movement from vacuoles to infected lysosomes (parasite indicated by oval shapes), (IIb) fusion of vacuole with
lysosome and intracellular particle realease.
pora sphaerica, which has been used in the past in
experimental biology for cell cycle synchronization.
Distinct differences in the inhibition of DNA-poly-
merase type I between mammalian and parasite cells,
revealed new interest in this unique structure as a
new antiparasitic lead. Besides its activity, the use of
aphidicolin is limited, because it is extremely fast
cleaved when given orally and its poor solubility has
stopped research in the past. By formulating aphidi-
colin as nanosuspension, these main disadvantages
can be minimized. In in vitro test systems with
infected RAW macrophages, the uptake of the
nanosuspensions improved the EC 50 value from 200
to 40 ng / ml in comparison to DMSO-dissolved drug.
The endocytic uptake of aphidicolin particles is
demonstrated microscopically (Fig. 7). The dark
spots display macrophages filled completely with
nanosuspensions (Fig. 7, middle), as visualised by
transmission electron microscopy (TEM) (Fig. 7,
right). In comparison, Fig. 7 (left) shows untreated
control of RAW macrophages seeded on Petri plates.
Compounds also approved for peroral use only
show a broad potential for i.v. or i.m. administration,
when formulated as nanosuspension. For example,
atovaquone is only for oral use, due to the fact that
 ¨
R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19 15

Fig. 7. Targeting of nanosuspensions to RAW-macrophages. Left: untreated RAW macrophages (TEM); middle: RAW-macrophages
incubated with 5% (V/ V) aphidicolin nanosuspension (circles) (light microscope); right: Leishmania-infected RAW-macrophages incubated
with 5% (v / v) aphidicolin nanosuspension (TEM). LM, lysosome membrane; NP, nanoparticle; P, parasite; VNP, nanoparticles in vacuole.

the poor solubility of micronized particles does not
allow i.v. application (capillary blocking). Formula-
tion of atovaquone as a nanosuspension for i.v. use
to treat murine toxoplasmosis showed a significant
reduction of Toxoplasma gondii at a dose of 0.3
mg / kg in comparison to 30 mg / kg when given
orally.
6. Large-scale production of nanosuspensions
The possibility and ability for large-scale pro-
duction of a delivery system or a dosage form is the
essential prerequisite for its introduction to the
pharmaceutical market. It is pointless to have a very
neat delivery system but no possibility to produce it
on a large scale to supply the market. In addition, the
production technology needs to be low-cost to
consider the financial restrictions of the health
systems, even in relatively rich countries. A technol-
ogy which is feasible but extremely expensive will
not find its way to the market, especially when
considering the recent discussions on the cost-to-
patient benefit ratios. In the mid-1980s in the UK,
calculations were made to multiply the medium
survival time of patients in years with a life quality
parameter. The resulting figure was compared to the
cost per operation. This means it was not considered
economical to perform a very cost-intensive heart
replacement for one patient resulting in a limited
quality of life compared to many low-cost opera-
tions, such as hip replacements, resulting in a high
number of survival years with a high quality of life.
Therefore, the cost factor for a technology is getting
more and more important.
The importance of the availability of large scale
production facility becomes evident when looking at
polymeric drug nanoparticles. For more than 30
years, intensive research has been performed in this
area, but this delivery system does not really exist on
the market. There is one product, Abdoscan  by
Nycomed; however, this is not a product for chronic
treatment of diseases but a diagnostic agent for
single administration (meaning a product with less
problems regarding regulatory acceptance). Depend-
ing on the type of nanoparticles, there are many
different reasons for the lack of products on the
market. However, from our point of view, a general
reason is the lack of a production method on a large
scale yielding a product of acceptable quality by the
regulatory authorities. An important point is that it is
not sufficient at all to have available a production
line which can produce on large scale. As a pre-
requisite for acceptance by the regulatory authorities,
this production line needs to be suitable for qualifica-
tion and validation. In addition, the technology needs
to be cost-effective. There are still problems to meet
all these requirements for the production of poly-
16 ¨
R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19

meric nanoparticles. In contrast to this drug,
nanoparticles can easily be produced on a large scale
using the high pressure homogenization technique
[50,35] already applied in various areas for large-
scale production.
High pressure homogenizers are available with
different capacities from a few hundred to a few
thousand liter per hour. The high pressure homogeni-
zation technology is well established in many differ-
ent areas of applications ranging from food to
lubricant production. In pharmaceutics, emulsions for
parenteral nutrition are produced by high pressure
homogenisation, which means production lines ac-
cepted for parenteral products are available.
For the production of a nanosuspension, the
macrosuspension needs to be passed somewhere
between three times up to five / ten or a maximum of
20 times through a high pressure homogeniser.
Instead of passing, say three times through one
homogenizer, three homogenizers can be placed in
series to produce the product in a continuous mode
(Fig. 8, upper). Using three homogenizers is possible
because the homogenizers are off-the-shelf equip-
ment and are in addition of low-cost. A suitable
homogenizer is the Rannie having a capacity of 1000
l at a maximum pressure of 1500 bar. To place
homogenizers in series is less economical where a
higher number of cycles is required. For example,
with five cycles and simultaneously a relatively small
batch size of, say 1000 l, a low-cost solution would
be to apply a discontinuous mode of one homogen-
izer. The suspension will be passed from the feeding
container through the homogenizer to the product
container, after this first cycle the suspension will be
passed back and so on, so that five passes could be
performed within 5 h of production (Fig. 8, middle).

Fig. 8. Scheme of production lines. Production of nanosuspensions applying three passes (cycles) by using three homogenizers (H) in series
(5discontinous production, upper), applying five to ten cycles by passing the product multiple times through one or two homogenizers
(5discontinous production, middle and lower; FC, feeding container; PC, product container).
 ¨
R.H. Muller et al. / Advanced Drug Delivery Reviews 47 (2001) 3 – 19
Where ten cycles are required, one could place two
homogenizers between the containers to keep the
production time constant at 5 h (Fig. 8, lower).
Applying pearl milling for the production of drug
nanoparticles requires milling times from hours to a
few days. Milling over a few days bears the risk of
microbiological problems, especially when perform-
ing the milling at room temperature or having
dispersion media providing nutrition to bacteria.
Production times of generally a few hours are,
therefore, a basic advantage of producing drug
nanoparticles by high pressure homogenization. In
addition, high pressure homogenization has a steriliz-
ing effect. It is a technique applied, for example, for
the rupture of cells including bacteria, thus increas-
ing the microbiological product quality.
Another important point is the contamination of
the product by erosion from the production equip-
ment. Pearl mills bear the risk of erosion of material
from the pearls — contamination of the product by
pearl material has been reported [51]. Even for oral
administration, eroded particulate material might
represent a problem. The formation of microparticles
from the pearls could occur, such microparticles
might interact with the M cells of the Peyer’s patches
with unknown consequences on the immune system.
Particle uptake by the M cells is also considered as
one approach for oral vaccination, therefore, distur-
bances of the immune system by continuous ad-
ministration of non-biodegradable microparticles
cannot be excluded. Such contamination does not
exist in products produced by high pressure homoge-
nization. Possible contamination of the nanosuspen-
sions could occur from metal ions coming off from
the wall of the homogenizer. The metal contamina-
tion was analyzed in nanosuspensions produced
under extreme conditions, i.e. 20 cycles at a maxi-
mum pressure of 1500 bar. The most dominant ion in
steel is iron, thus this metal was analyzed in the
nanosuspensions and was found to be below 1 ppm
[52], i.e. an uncritical level.
7. Perspectives
Drug nanocrystals — independent of their way of
production — represent a technology to overcome
solubility problems and bioavailability problems of
drugs which can be generally applied to all poorly
17
soluble drugs. Any drug can be transformed to drug
nanoparticles leading to an increase in saturation
solubility, dissolution velocity, and providing the
general feature of an increased adhesiveness to
surfaces. Surface modification of the drug nanocryst-
als can further increase the benefits, e.g. by produc-
ing mucoadhesive nanosuspensions for oral applica-
tion or surface-modified site-specific nanoparticles
for intravenous injection (e. g. targeting to the brain,
bone marrow etc.). A further advantage is that this
innovative nanosuspension technology can be com-
bined with traditional dosage forms, e.g. incorporat-
ing drug nanoparticles into pellets or tablets for oral
delivery. An outstanding feature of the technology is
its simplicity. The more simple the system is, the
higher are the chances to launch products on the
market. The success of drug nanoparticles, especially
the third generation product nanosuspensions, will be
demonstrated by the number of products on the
market in the near future.
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