Stability issue of nanosuspensions in drug delivery

Yancai Wang, Ling Zhang, Qiwei Wang, Dianrui Zhang

PII: S0168-3659(13)00463-X
DOI: doi: 10.1016/j.jconrel.2013.08.006
Reference: COREL 6834

To appear in: Journal of Controlled Release

Received date: 4 May 2013

Accepted date: 6 August 2013

Please cite this article as: Yancai Wang, Ling Zhang, Qiwei Wang, Dianrui Zhang,
Stability issue of nanosuspensions in drug delivery, Journal of Controlled Release (2013),
doi: 10.1016/j.jconrel.2013.08.006

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 ACCEPTED MANUSCRIPT

Stability issue of nanosuspensions in drug delivery

Yancai Wanga,*, Ling Zhangb, Qiwei Wangc, Dianrui Zhangc

a
School of Chemistry and Pharmaceutical Engineering, Shandong Polytechnic University, Jinan

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250353, PR China

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b
Library, Shandong University of Finance and Economics, Jinan 250014, PR China
c
College of Pharmacy, Shandong University, Jinan 250012, PR China

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*To whom correspondence should be addressed:
Yancai Wang, Ph.D.

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School of Chemistry and Pharmaceutical Engineering, Shandong Polytechnic University, Jinan
250353, China
Tel/ fax: +86 531 89631208
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E-mail address: [email protected] (Y. Wang)

ABSTRACT:
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Nanosuspensions are nanosized colloidal dispersion systems stabilized by surfactants, polymers or

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a combination of both. Due to nanosizing results in the creation of new interfaces and in a positive
Gibbs free energy change, nanosuspension is a thermodynamically unstable system with tendency
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of agglomeration or crystal growth. Despite the nanosuspensions technology has been extensively
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investigated, stability issue is still the limitation and shortcoming for its application on
pharmaceutical industrial. Furthermore, the knowledge on empirical relationship between the
stabilizer efficacy and stability of the nanosuspension is relatively deficiency. This review
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especially focused on the stability issue of nanosuspensions in drug delivery to retrieve the state
art of the nanosuspensions. Therefore, the main contents of present review including the
presentation of nanosuspensions instable, the method and guideline for section and optimizing
stabilizers, the approaches for enhancing stability, as well as the other influencing factors on the
stability of the prepared nanosuspensions. For a given drug candidate having a set of properties,
this article could be used as a reference in making educated choice of stabilizer and in optimizing
operation parameters for nanosuspensions formulation, rather than a trial and error approach that
is being practiced currently.
Keywords: Nanosuspensions; Stability; Stabilizers; Aggregation; Crystalline transformation

Contents
1. Introduction ................................................................................................................................... 2
2. The common stability issues of nanosuspensions ......................................................................... 4

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2.1. Aggregation ........................................................................................................................ 4

2.2. Sedimentation ..................................................................................................................... 6
2.3. Crystalline transformation ................................................................................................. 7
2.4. The plasma stability ........................................................................................................... 8
2.5. The chemical stability ...................................................................................................... 10

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3. Guidelines for stabilizers selection and usage in nanosuspensions preparing............................. 11
3.1. The conventionally used stabilizers.................................................................................. 11

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3.2. The influence of logP and enthalpy of the candidate drug ............................................... 13
3.3. The surface energy and specific interactions ................................................................... 14

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3.4. The hydrophobic moieties of the stabilizers ..................................................................... 17

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3.5. The optimized concentration and temperature for the given stabilizers .......................... 18
3.6. The pH environments for optimizing stabilizers functions ............................................... 20
3.7. The drugs should have little solubility in the stabilizers solution .................................... 21

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3.8. The influence of stabilizers on morphological structure of the nanocrystals ................... 22
3.9. The influence of stabilizers on nanocrystals particle size ................................................ 23
3.10. The influence of stabilizers on zeta potential ................................................................. 24
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4. The influences of preparation approach on stability ................................................................... 25
4.1. High pressure homogenization approach......................................................................... 25
4.2. Media milling approach ................................................................................................... 28
4.3. Microfluidizer approach................................................................................................... 29
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4.4. Bottom-up approach......................................................................................................... 30

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5. The solidification methods for improving nanosuspensions stability ......................................... 31

5.1. Spray drying ..................................................................................................................... 31
5.2. Freeze-drying ................................................................................................................... 32
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5.3. Hydrogels ......................................................................................................................... 34

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6. Challenges and future perspectives ............................................................................................. 35

References ....................................................................................................................................... 36
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1. Introduction

Due to water insoluble thereby resulted in non-optimal physicochemical and

biopharmaceutical properties, more than half of all new drug candidates entering the development
pipeline failed currently [1]. Drugs nanosizing, such as nanosuspensions, represent an interesting
and potentially very useful method to improve the bioavailability of the hydrophobic drugs [2].
Nanosuspensions are defined as liquid unique sub-micron colloidal dispersions of nanosized pure
drug particles stabilized by suitable polymer and/or surfactant with particle size in the range
1-1000 nm [3]. The formulation of active pharmaceutical ingredients (APIs) as nanosuspensions
has gained popularity in the pharmaceutical industry in the last 10 years, and a few
nanosuspensions products are already on the market and many more novel nanosuspensions are
being extensively designed and studied by the academia and industry [4].

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Nanosuspensions platform is an efficient and intelligent drug delivery system for water
insoluble drugs, as the saturation solubility and the surface area available for dissolution increased
[5, 6]. Generally, the biopharmaceutical advantages of water insoluble drugs formulated as
nanosuspensions including improvement in formulation performance, such as high drug loading,

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reproducibility of oral absorption, improved dose-bioavailability proportionality, reduced toxicity

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and side effects, and increased patient compliance via reduction of number of oral units to be
taken [7, 8]. Furthermore, this technology is very interesting also because of the little use of

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excipients with respect to drugs, thereby reducing the risk of toxicity from these components [9,

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10]. The high ratio of the carried drugs to excipients is particularly advantaged for
nanosuspensions designed to target cancer cells, especially if a large amount of drugs is delivered

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for each targeting event [11].
Despite the progress of the knowledge in this field, nanosuspensions have the drawbacks of
instability caused by nucleation and particle growth [12]. The high surface area of drug
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nanosuspensions, by the virtue of which they exhibit their unique biopharmaceutical
characteristics, also renders them thermodynamically unstable and promotes agglomeration and
crystal growth [13]. Therefore, the instability is the most disadvantages of the nanosuspensions.
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And the stability issue is the inevitable encountered problems in the nanosuspensions technology
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developments and in the pharmaceutics industrial application. Therefore, it’s also the limitation
step in this platform development [14, 15]. The high surface energy of nano-sized crystals resulted
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in particle size growth, a phenomenon known as Ostwald ripening, in the absence of appropriate
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stabilizers. Flocculation or crystal growth during the production process or shelf life of the
nanosuspensions may directly impact dissolution and in vivo performance due to the formation of
larger particles with a reduced specific surface area [16].
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Currently, although there are several articles related to nanosuspensions, the nanosuspensions
technique suffers from a lack of systematic understanding about the requirements of the stabilizers
and there was still little known about the stability of the nanosuspensions. Stabilizers are vital to
prevent aggregation of high-energy nanosuspensions [17]. However, different drugs requiring
different polymeric stabilizers and it are unrealistic to find a single stabilizers system that works
for all different drugs and preparing processes [18]. Unfortunately, there was not a systematically
empirical or theory guideline for the stabilizer selection and optimizing. And there is still little
empirical known about the relationship between the stabilizer efficacy and stability of the
nanosuspensions. Thus, to find a proper stabilizer for a given formulation is often difficult. As a
consequence, currently, a scientific rationale stabilizers selection for a specific formulation is often
by a simple trial-and error approach [19]. This is a time-consuming effort required manner with
low efficiency. Furthermore, the type and amount of stabilizer also has a significant effect on the

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physical stability and the in vivo behaviors of the nanosuspensions [20, 21].
Beside above mentioned problems, the potential raised toxicity concerns if the stabilizers
used in large quantity for a long term, also limited the therapeutic application of the drug
nanosuspensions [22]. For example, Tween® 80, a commercial surfactant that widely used

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nanosuspensions preparing, could cause serious neuro- and nephrotoxicity as well as acute

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hypersensitivity reaction [23].
To assist in stabilizers selecting, a few studies focused on the developing empirical

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relationships between stabilizer efficacy and the properties of the drug compounds have been

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published recently [24]. The present review aimed to summarize the methods for improving the
stability issue of the nanosuspensions and to establish a general guide for the stable

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nanosuspensions preparation protocol. And to induction general stabilizers selecting directions for
the nanosuspensions formulation also is a mission of this review. Therefore, beside the instability
presentation of nanosuspensions and the guidelines for optimizing the stabilizers, the effects of
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nanosuspensions preparing approaches and solidification process of prepared nanosuspensions on
the stability also will be discussed in this article. And it could be used as a general guideline for a
stable nanosuspensions preparing.
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2. The common stability issues of nanosuspensions

2.1. Aggregation
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The nanosuspensions are not stabilized permanently by inappropriate stabilizers and

aggregation may occur during storage or being solidification process, although there are several
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advantages on nanosuspensions formulations. The unsuitable stabilizers resulted in the

agglomeration of smaller particles in the nanosuspensions due to the Ostwald ripening
phenomenon [25]. Ostwald ripening is a phenomenon whereby coarse particles grow at the
expense of fine particles re-dissolving [26]. In other words, the smaller particles sized
nanocrystals are more soluble than large ones, mass transfer occurred from the fines to the coarse
particles [27].
To reduce aggregation and Ostwald ripening, the formulated drugs should poorly soluble
anyway thus leading to only very little changes in the dissolved concentration during the
nanosuspensions preparation. Furthermore, the particle size of the prepared nanosuspensions
should be relatively homogenous in size thus avoiding larger differences in the saturation
solubility between differently sized crystals [28]. However, the nanosuspension is a
thermodynamics instable colloid disperser system. Therefore, aggregation is an inherent property
of the nanosuspensions, which is owned to the Ostwald ripening and the tendency of the nanosized

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system to reduce the Gibbs free energy [29].

Thereby, how to limit or restrain this aggregation is an important task during the
nanosuspensions developments. The use of a proper stabilizer is required for the stable
nanosuspensions preparation. The efficiency of a particular stabilizer depends on its potential for

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interaction with the drug compound [30]. Steric and ionic stabilization are the two most common

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stabilizers that are used for a stable nanosuspensions preparing.
The aggregation may be occurred during either the preparation process or the shelf-time. For

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the top-down approach on nanosuspensions produce, as the increase of surface area, aggregation

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of nano-sized drug particles occurred due to the thermodynamic effect, ultimately reducing the
efficiency of the process [31]. Thus, the use of a stabilizer is very important as it is needed to

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cover the surface of nanoparticles during milling or high pressure homogenizing. During the
storage, the aggregation becomes a more importantly stability issue. Appropriate stabilizers and
concentration selected may be the critical parameters for the stability [32]. The amphiphilic
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polymer, such as TPGS, could be adsorbed onto the surface of nanosuspensions particles. And the
adsorbed chain molecules on surface have ceaseless thermal motion, resulting in dynamically
rough surface preventing coalescence by repulsive entropic force. For example, in the NVS-102
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nanosuspensions, levels of d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS 1000) at
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4:1 (drug:TPGS 1000) ratio had shown significant growth of nanocrystals during the stability
study at the 6 week time point. However, at 2:1 and 1:1 (drug:TPGS 1000) ratios proved to be
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capable of producing more stable nanosuspensions with respect to particle size, demonstrating
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minimum aggregation or crystal growth during storage. Additionally hydroxypropyl

methylcellulose (HPMC) 3 cps produced synergistic stability effects with TPGS 1000 for
inhibiting aggregation [16]. The suspending agent, such as HPMC, commonly used in suspension
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formulating, could be able to increase the viscosity of the nanosuspensions and reduce the
sedimentation rate. Furthermore, it also could to form stereospecific blockade between the
nanosuspensions particles and inhibited the particles contacting.

It was reported that functional copolymers bearing alkynyl or azido groups which may be

prevent the aggregation by “click-chemistry” mediated cross-linking [2]. The monomer and

polymer were synthesized and 1,3-dipolar cycloaddition of the polymers were successfully in

solution and on the surface of nanocrystals. In that report, paclitaxel nanosuspensions (100 nm)

were prepared by wet milling and stabilized by a tailored copolymeric surfactant composed of

methoxy-poly(ethylene glycol) -b -poly (ε-caprolactone) (mPEG-b-PCL), which bears pendant

alkynyl groups incorporated in the hydrophobic PCL block. These functional groups were then

reacted in a copper-catalyzed 1,3-dipolar cycloaddition reaction with azido-groups of a

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cross-linking polymer. Diblock mPEG-b-PCL copolymers were chosen because mPEG will

prevent aggregation through steric hindrance and because the more hydrophobic PCL would

sequester to the hydrophobic surface of the nanosuspensions and provide an anchoring point for

the polymer. The successful cross-linking of the polymers around the drug nanosuspensions may

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serve as a powerful tool to prevent aggregation and control dissolution rate. Thus, it was

hypothesized that the cross-linked polymer coating could help to stabilize the drug

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nanosuspensions and to avoid aggregation.

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2.2. Sedimentation

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Nanosuspensions belonged to colloidal dispersions. Compared with true solutions where
molecular dispersions are homogeneous, colloidal dispersions are intermediate in size between
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true solutions and coarse dispersions. Sedimentation is the extremity presentation of instable
phenomenon of the nanosuspensions [33]. Generally, the aggregation and the Ostwald ripening is
the primary step of instability. The transition of size ranges, either from molecular dispersions to
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colloids or from colloids to coarse dispersions, is very gradual. However, when the gravity of the
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drug particle greater than buoyancy force of dispersion system provided, the sedimentation will be
occurred inevitable. This process is irreversible and un-redispersible. Therefore, how to predict
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and prevent the sedimentation is the chief consideration in nanosuspensions formulating [34]. For
example, ciclosporin A (CsA) nanosuspensions prepared with various polymers using a media
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milling method, PVA stabilized nanosuspensions showed no creaming or sedimentation

phenomena and gave the smallest particles of about 530 nm [35].
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Beside the stabilizers type which may be induced the sedimentation of nanosuspensions, the
other factors also the potential reasons for incurs this instability. Acetaminophen nanosuspension
was prepared using nanoprecipitation in microfluidic reactors [36]. Four variables, namely
concentration of surfactant, solvent and anti-solvent flow rate and solvent temperature were used
as input variables and time of sedimentation of nanosuspensions was considered as output variable.
It was revealed that anti-solvent flow rate and temperature have direct relation with time of
sedimentation, whereas solvent flow rate generally has reverse relation with the time of
sedimentation.

Flocculation, a type of sedimentation, is an out-of equilibrium phenomenon of a colloidal

nanosuspension. And it results from attractive interactions between particles, and its kinetics can

be quantitatively related to the so-called DLVO potential. Flocculation of nanosuspensions may

occur by polymer bridging, charge neutralization, polymer-particle surface complex formation or

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depletion flocculation, or by a combination of these mechanisms [37]. Depending on the nature of

the polymer and the character of its interaction with the nanocrystals surface, there are two main

possible mechanisms of flocculation: (1) bridging as a result of adsorption of a macromolecule

onto the surface of nanocrystals and (2) surface charge neutralization [38]. In very dilute systems,

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flocculation generates flocs with a fractal structure, which can be modeled using numerical

simulation or measured using static single light scattering. For more concentrated

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suspensions-particle volume fraction being between 0.1 and 50% or above-the flocs grow and

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percolate, leading to the formation of a gel-like structure.

Flocculation also could be used as an intermediate step for preparing wettable nanocrystals

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powders with high drug loading and rapid dissolution [39]. Naproxen nanosuspensions, prepared
by anti-solvent precipitation, were flocculated with sodium sulfate, filtered, and dried to form
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redispersible powders for oral delivery by Chen et al [40]. The nanosuspensions were stabilized by
PVP K15 and Pluronic F127. The filtration process increased the drug loading by up to 61%
relative to the initial value, as unbound surfactant was removed with the filtrate. The particle size
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of the redispersed dried powder was comparable to the original value in the aqueous
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nanosuspensions prior to flocculation, and consistent with primary particle sizes observed by
scanning electron microscopy (SEM). The redispersion ability of the dried powders was examined
as a function of the salt concentration used for flocculation and the surfactant composition and
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concentration. In concluding, flocculation followed by filtration and drying is an efficient and

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highly reproducible process for the rapid recovery of drug nanocrystals to produce wettable
powders with high drug loading and rapid dissolution.
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2.3. Crystalline transformation

The solidification process, such as freeze-drying and spray drying, is the common approach
for further improving the physical and chemical stability of the nanosuspensions [41]. However,
during the solidification operating, the minimal energy is required for forming the amorphous,
noncrystalline forms [42]. In other words, the formation of the amorphous form requires much less
energy than crystal formation. Generally, fully or part amorphous state of the insoluble drugs in
nanocrystals is theoretically an ideal method to furtherer improve the dissolution rate [4].
However, molecules of nanocrystals in the amorphous state are thermodynamically unstable
relative to the crystalline state. Therefore, although the amorphous form of the formulated
nanosuspensions has higher solubility and a higher dissolution rate than the crystalline state,
amorphous solids are thermodynamically instable and tend to transform to a crystalline state,
which limits their extensively commercialization [11]. Therefore, a crystalline nanosuspensions

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drug is more stable and thereby often more favorable [43]. What's worse, there was a deficiency in
appropriate controlling methods for this transformation. Therefore, once the production process
generates amorphous material, there is a risk of crystalline transformation during storage or
release and thereby the crystalline transformation is an inevitable problem of encountered in the

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storage stage of the nanosuspensions.

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The crystalline transformation will be induced solubility and thereby therapeutic effects
alteration [44]. For a compound of nanosuspensions formulated with polymorphism, the risk exists

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that the APIs crystalline transformation between crystallize and amorphism during storage [45].

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Besides the losing of beneficial properties (high solubility and high dissolution rate), the
transformation of the formulation also induced instability problem. Furthermore, this

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transformation may be related to the bioactivity changes of the formulated drugs in clinical usage,
which is a potential incentive adverse reaction. Therefore, how to monitor and tight control of the
nanocrystals crystallinity during the solidification production process is an important task for a
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formulation scientist [4]. For example, polymer polyvinylpyrrolidone (PVP) could decreasing
bicalutamide crystallization rate significantly in a system with PVP concentrations as low as
0.01% (w/w), without changing the polymorph formed. And the crystal habit could be altered
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already at PVP concentrations as low as 0.001% (w/w). Therefore, it could be to monitor the
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crystalline transformation during the freeze-drying by real-time X-ray Diffraction. For more detail
information, the readers could be referred to some drug crystals articles [46-48].
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It was reported that the freezing rate as well as the water/ tertiary butyl alcohol (TBA) ratio
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of the fenofibrate nanosuspensions before freeze drying could be used to control the crystallization
of the drug [49]. When the solutions were frozen rapidly or when the fraction water was decreased,
the crystal size was decreased and the dissolution rate was strongly increased (Fig. 1). The
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prepared nanosuspension was a dispersion containing 30% w/w fenofibrate in mannitol, followed
by freeze-drying. The crystallization of fenofibrate and mannitol could occur during freezing
and/or drying. If the crystallization occurred during freezing, faster freezing or a smaller water
content of the solution resulted in a higher nucleation rate. Subsequently, more nuclei were formed
resulted in more crystals, which were consequently smaller. If the crystallization process
preformed during drying, the temperature was above the glass transition temperature of the
maximally freeze-concentrated fraction (Tg′) and under the eutectic temperature (Te). And the
growth of the nanocrystals is limited to the size of the interstitial spaces between the solvent
crystals.

2.4. The plasma stability

Beside the nanosuspensions could be administrated by oral dosage forms, the intravenous

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delivery also is an important approaches for the water insoluble compounds formulated

nanosuspensions [50, 51]. Therefore, the plasma compatibility of the developed nanosuspensions

is a critical issue. It is clear that a critical characteristic of nanosuspensions formulations is their

ability to alter pharmacokinetics and, in particular, to demonstrate long-circulating properties, in

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order to achieve passive drug targeting to disease sites characterized by leaky vasculature via the

enhanced permeation and retention (EPR) effect. Thus, following intravenous administration, the

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nanosuspensions must retain good physical stability properties, maintaining reasonable structural

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integrity, providing a greater drug half-life and increasing drug exposure (AUC) compared to free

drug. In our opinion, all the factors, including but not limited to, particle size distribution, surface

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charge, morphology, and surface hydrophilisity/hydrophobisity, as well as the infusing rate and

concentration, are the potential reason for incurring instability of the nanosuspensions, as all these
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factors are dominating the in vitro and in vivo stability of the nanosuspensions. Among of all the

reasons, the surface charge and surface hydrophilisity/hydrophobisity may be the main influencing
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factor for causing the instability in plasma. When the nanosuspensions infused into the plasma, the
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environment of the nanosuspensions changed significantly, such as pH, ionic strength, et al, which

could be alter the surface charge and zeta potential of the nanosuspensions and induce the
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aggregation of the nanosuspensions. However, there was still no special article published focused
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on the in vivo stability influencing factors and we are planning to conduct some research on this

issues.
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A relative study conducted by Aramwit et al evaluated the in vitro plasma compatibility of a

formulated nanosuspension under conditions that mimic intravenous injection [52]. In that study,
the lyophilized nanosuspension formulation was reconstituted with sterile water for injection,
followed the drug was then further diluted with 0.9 % w/v NaCl or 5% w/v dextrose to make
concentrations of 1.5 and 5.0 mg/ml with each diluents. To simulate different intravenous infusion
rates, 1.5 mg/ml redispersed nanosuspensions were diluted with plasma at the ratio of 1:1 and 1:2,
and the 5.0 mg/ml redispersed nanosuspensions were diluted with plasma at the ratio of 1:2 and
1:10. The particle aggregation is the main instability of the nanosuspensions. Therefore, the
influencing factors, such as the types of diluents, the formulation/plasma ratio dependent upon
infusion rate, the plasma flow rate, and the incubation time were evaluated with respect to particle
aggregation. It was found that aggregation occurred very rapidly in all conditions and that more
aggregation taken place with higher drug concentrations in the mixture with plasma. It was also
revealed that there was minimum aggregation at the concentration of 1.5 mg/ml when delivered at

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an infusion rate of 2.5 ml/min. This combination is recommended for further toxicological and
clinical evaluation.

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Fig. 1. SEM of crystallized fenofibrate nanosuspensions prepared at different freezing rates. All
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dispersions contain 30% w/w fenofibrate in mannitol. (Magnification left column 1500×;
magnification right column 10,000×) (adopted from [49]).
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2.5. The chemical stability

Beside the physical stability of the nanosuspensions mentioned above, the chemical stability
is also an important aspect for the drug delivery platforms in the pharmaceutical science [53].
Generally, the nanosuspensions were prepared in a dispersion medium of water or water mixture
environments, although there was a few process operated in non-aqueous mediums [54]. Therefore,
the chemical stability, such as hydrolyses and oxidation, is a considerable problem in the
nanosuspensions formulating. And this is the shortage of the nanosuspensions on drug chemical
stability [55]. However, it’s also the potential approach for improving the chemical stability for
chemical labile drugs, as the final solid and concentrated state protected the drug from the hazard
of photoallergy, hydrolyses as well as oxidation. But for hydrolyses labile compounds, how to
process the nanosuspensions preparation with chemical stable is a tough and troublesome
question.

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It was reported that a remarkable improvement in chemical and photo-stability of quercetin

molecules in nanosuspensions was observed compared with the solution stored in the same
conditions [56]. The increased chemical stability could not only hold the activity but also reduce
the toxicity of quercetin. For the improved stability, it could be explained by the following reasons.

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The molecules of the stabilizers covering on the surface of the nanocrystals could shield the inner

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compound from the oxygen and light. Thereby, for the nanosuspensions only the particle surface
uncovered by the stabilizer was accessible to water and light, which means there would be the

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formation of a degraded outer monolayer of molecules protecting the inner part of the drug

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nanoparticles (similar to oxidized layer on top of aluminium). Due to the protection of the
stabilizer layer, even in this outer layer only a few molecules degraded. Thus, nanosuspensions

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technology could be used as an effective approach to improve the stabilization of the chemical
labile drugs, such as quercetin and curcumin.
Omeprazole, a proton pump inhibitor, is a poorly soluble and chemically labile drug with
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high degradation rate in aqueous media. Moschwitzer et al formulated the omeprazole as
nanosuspensions [28]. The HPLC analysis proved the predominance chemical stability of the
nanosuspensions produced by high pressure homogenization (HPH) in comparison to an aqueous
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solution. Even 1 month after production no discoloration or drug loss was recognizable when the
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nanosuspension was storaged at 0 °C.

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3. Guidelines for stabilizers selection and usage in nanosuspensions preparing

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3.1. The conventionally used stabilizers

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Conventionally, stabilizers are indispensable in the nanosuspension formulations to prevent

them from aggregation and agglomeration. Stabilizers commonly used to stabilize
nanosuspensions including polymers (such as polyvinyl pyrrolidone (PVP), hydroxypropyl methyl
cellulose (HPMCs) [57], and hydroxypropyl cellulose (HPCs) [58], d-α-tocopherol polyethylene
glycol 1000 succinate (TPGS 1000) [27, 39, 59], polyethylene glycols (PEGs) [60], polyvinyl
alcohols (PVAs) [24], amphiphilic amino acid copolymers [18], crystalline cellulose [61]), ionic
surfactants (e.g., sodium dodecyl sulfate (SDS), Sodium lauryl sulfate (SLS) [58], Poly(ethylene
imine) (PEI) [62], chitosan [63]), and nonionic surfactants (e.g., Tweens and block copolymers of
poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO-PEO) [64], also known as Pluronics®).
Beside these frequently used stabilizers, it was reported that the food proteins, such as soybean
protein isolate, whey protein isolate and β-lactoglobulin, also could be used as stabilizers in the
nanosuspensions formulations. Ionic surfactants stabilize suspensions via electrostatic repulsion,
while polymers and nonionic surfactants facilitate suspension stability via steric repulsion [19].

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The stabilizers used in the preparation of nanosuspensions should adsorb onto the surfaces of

drug nanosuspensions and provide steric or electrostatic stabilization effect, powered by the force

of hydrophobic moieties in the stabilizers. Thus, the surface of nanosuspensions particles could be

covered by the adsorbed stabilizers. The adsorbed chain molecules on surface have ceaseless

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thermal motion, resulting in dynamically rough surface preventing coalescence by repulsive

entropic force [24]. When the absorption procedure completed, there may be forming hydrogen

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bonding and electrostatic interactions, which could be further strengthen the absorption. When the

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hydrogen bonds in the nanosuspension system formed, there will be a strong electrostatic

repulsion between the totally ionized functional groups of macromolecules and the negatively

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charged surface of the adsorbent. For establishing successful stabilization effects within a

reasonable processing time, strong and fast adsorption at full coverage and a long time scale for
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desorption is necessary in addition to steric repulsion [65]. The higher the molecular weight of a

steric stabilizer, the slower adsorption rate is observed [27]. Polymer length and molecular weight
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of a polymer acts as the thermodynamic driving force for the physical adsorption on the surface of
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the particle.

The surfactant F127 is one of the least toxic, commercially available, block copolymers [66].
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And it has been extensively used in a variety of pharmaceutical formulations including low
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molecular mass drugs and polypeptides. Generally, Polymer type steric stabilization is good for
the stability of drugs during processing since it does not usually destroy the crystal structure of
drug particles unlike the conventional surfactants of small molecular weights such as sodium
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lauryl sulfate (SLS) [65]. The surfactants tend to form micelles containing a small number of
dissolved drug molecules.
For the top-down nanosuspensions preparing approach, the particle size of a drug usually
decreases to a steady state value with time, which depends on the kind of stabilizers selected.
Furthermore, the stability and robustness of nanosuspensions are mainly manipulated by various
formulation and process variables [67]. Therefore, the stabilization effect of the stabilizers is a
critical parameter and consequently the selection of proper steric and electrostatic stabilizer and
optimum its quantity plays a major role in the nanosuspensions formulating. Based on the
characteristics of nanosuspensions, developing and discovering new stabilizers also is an
important aspect for nanosuspensions technology [68].
As a high nutritional values and generally recognized as safe candidate stabilizers, food
biopolymers, especially food proteins, are widely used in formulated foods [69]. He et al prepared

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indomethacin nanosuspensions using the three food proteins (whey protein isolate (WPI), soybean
protein isolate (SPI) and β-lactoglobulin (β-lg)) as novel stabilizers by precipitation-
ultrasonication method [70]. It was found that the proteins could adsorb onto the surface of drug
particles through interaction between the hydrophobic areas of the proteins and the drug. The

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mean particle size of the initial nanosuspensions and that after 15 day storage at 4 °C was

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393.3/404.4 nm, 281.8/289.8 nm and 285.6/286.5 nm with bimodal distribution for SPI, WPI, β-lg,
respectively. This indicated a good stability of the three stabilizers formulated nanosuspensions.

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To further improving the stability of the nanosuspensions, employing another polymer served

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as an auxiliary stabilizer is feasible. For example, the physical stability of nitrendipine
nanosuspensions was remarkably improved by the chitosan modification [63]. Chitosan is a

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cationic polysaccharide. Compared with the PVA stabilized nitrendipine nanosuspensions, the
stability of the chitosan modified nanosuspensions increased significantly in 30 °C for 24 days.
Electrostatic repulsion and steric stabilization are the two main mechanisms for chitosan
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improving the nanosuspensions stability. The zeta potential of PVA prepared nanosuspensions was
only -6.58 mV which is far below the minimum value to keep the particle size constant. However,
the zeta potential of the chitosan modified nanosuspensions was about +40 mV. Therefore, the
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enhanced electrostatic repulsion prevented the growing of the particle size. Furthermore, the
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chitosan provide steric stabilization effect by deposited on the surface of the nanosuspensions
further increased the particle size stability of the prepared nanosuspensions.
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3.2. The influence of logP and enthalpy of the candidate drug

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A study conducted by George et al revealed that the logP and enthalpy of candidate APIs
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possessing a direct correlation on the feasibility of formation of a stable nanosuspensions [71].

The APIs with low enthalpy values were observed to be poor candidates for formulation as
nanosuspensions irrespective of the stabilizer used. The APIs with low logP values were formed
the least stable nanosuspensions. George et al and coworkers selected six drug molecules with
different molecular weight, melting point, solubility, logP, pKa, enthalpy and morphology, and
four stabilizers with a range of physical properties to reveal the influence of logP and enthalpy on
nanosuspensions stability. The six drug was Naproxen, A, B, C, D and E, and the physical
properties were shown in Table 1. The four stabilizers were SLS, DOSS, TPGS and Pluronic F127.
The logP and enthalpy of candidate APIs seemed to have a direct relationship with the feasibility
of forming a successful nanosuspension by both steric and electrostatic stabilization. As shown in
Fig. 2, the most likely candidate for media milling was a drug substance with a high enthalpy and
hydrophobicity which can be stabilized either electrostatically or sterically. The least likely
candidate will be one that is hydrophilic and having a very low enthalpy value.

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Fig. 2. Trend analysis of particle size stability at the end of 6 week storage at 4 °C with respect to
enthalpy and LogP of the six drug candidates. And the proposed generic formulation of
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nanosuspensions based on drug properties (adopted from [71]).
Table 1. The physical properties of the six drugs candidates.
Drug M.P. ( °C) Enthalpy (J/g) Solubility logP Mol.
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(μg/mL) weight
Naproxen 153.0 244.9 31 3.2 230.26
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A 263.0 88.9 3.5 3.0 388.4

B 284.7 114.7 170 1.6 377.43
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C 158.0 160.6 5 2.7 293.72

D 241.6 74.0 74 1.7 419.46
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E 151.4 80.0 0.3 4.5 448.56

3.3. The surface energy and specific interactions

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Stabilizer, covering the surface of nanosuspensions producing ionic or steric stabilization, is

an essential component of the formulations. The hydrophobic chain adsorbed on surface of the
nanosuspensions having ceaseless thermal motion, resulted in dynamically rough surface
preventing coalescence by repulsive entropic force. However, the stabilizers diffusion, convection,
adsorption, and desorption occurred simultaneously with the mechanical fracturing and new
surface area generating of the nanosuspensions system [35]. This is a dynamic state. Therefore,
there exiting a surface area rate competing between the stabilizers net adsorbed surface and the
new generated surface. The higher surface adsorption rating produced the higher efficiency of the
preparing methods and the stability of the prepared nanosuspensions. Thus, to illustrate which
factors dominating the net adsorption rating is a mission of relative studies.
The stabilization ability of the stabilizers and the subsequent steady state particle size of
drug nanocrystals depend on various parameters including surface energy and specific interactions.

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However, the interaction between stabilizers and drugs was a complicated function of various
parameters including surface energy and the existence of functional groups. The similarly surface
energy between the stabilizer and nanocrystals is a critical factor for determine the stabilization
efficiency. Choi and his coworkers studied the nature of interactions between polymeric stabilizers

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and drugs by analyzing the steady state particle sizes of 7 drugs (Ibuprofen, Naproxen,

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Prednisolone acetate, Nifedipin, Hydrocortisone acetate, Itraconazole, Anthracene) obtained by
wet comminution [65]. The stabilization ability of two typical pharmaceutical polymers, HPC and

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PVP was examined based on the steady state particle sizes of the seven different drugs produced

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nanosuspensions. The surface energy of drugs and polymers was exhibited in Table 2. and was
found to be an important factor, though it could not solely determine the steady state values of the

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nanosuspensions particle size. In the HPC cases, only the two drugs, prednisolone acetate and
hydrocortisone acetate, show no significant size reduction. Interestingly, only the two drugs have
hydroxyl functional groups (-OH), which HPC also has. Therefore, specific interactions such as
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hydrogen bonding between two hydroxyl groups exist in the two cases. In the PVP cases, only
anthracene shows unexpected behavior. PVP cannot have any strong hydrogen bonding, and
anthracene has no polar functional group at all, while all the other drugs have more than one polar
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group. If the specific interactions between functional groups play a role, the results of Table 2
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suggested that they tend to interfere the stabilization activity of polymers. Hydrophobic surface
without having polar functional group seems to be ideal for HPC and PVP to physically adsorb
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and produce steric stabilization.

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Table 2. Relation between surface energies (in parenthesis) and the mean particle sizes of drug
nanosuspensions processed by wet comminution process in the presence of HPC and PVP [65].
Mean particle size (standard deviation) (nm) HPC (48 dyn/cm2) PVP (42 dyn/cm2)
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Ibuprofen (52 dyn/cm2) 420 (±80) >10 K

Naproxen (42 dyn/cm2) 120 (±40) 210 (±80)
Prednisolone acetate (39 dyn/cm2) >10 K 1460 (±1590)
Nifedipin (38 dyn/cm2) 190 (±70) 2290 (±1900)
Hydrocortisone acetate (37 dyn/cm2) >10 K 3850 (±3190)
Itraconazole (36 dyn/cm2) 310 (±130) 8240 (±13,000)
Anthracene (33 dyn/cm2) 70 (±40) 210 (±130)
Similarly, Verma et al employed the Atomic force microscopy (AFM) for the first time to
demonstrate direct evidence of the role of the characteristics of the adsorbed stabilizers (Pluronic
F68, Pluronic F127, PVP K30, HPC) layer on ibuprofen nanosuspensions stabilization and to
elucidate the arrangement/conformation of the stabilizer molecules on the ibuprofen nanocrystals
surface [19]. In addition, the adhesion forces between the AFM tip and the adsorbed stabilizer was
also investigated to obtain an understanding of the nature of the interactions involved in the
adsorption process. As illustrated in Fig. 3, the root-mean-square (rms) roughness of the bare

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ibuprofen surface height was 14.0 nm. The morphology and arrangement of the HPMC adsorbed
on the ibuprofen are completely uncoiled and are adsorbed in an open extended conformation on
the ibuprofen surface. The height of the chains was approximately 2.7 nm, while the width of the
chains is measures 43 nm. PVP adsorbed in globule-like structures with heights ranging from 12

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to 28 nm and widths between 100 and 200 nm.

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Fig. 3. AFM height image of the ibuprofen surface adsorbed by different stabilizers. A is the bare
ibuprofen surface. B is the HPC adsorbed on ibuprofen surface. C is the PVP adsorbed on
ibuprofen surface. D is the HPMC adsorbed on ibuprofen surface. E is the Pluronic F68 adsorbed
on ibuprofen surface. F is the Pluronic F127 adsorbed on ibuprofen surface (adopted from [19]).
In addition, preferential adsorption can be observed on certain faces of the ibuprofen crystal
due to crystallographic anisotropy. Pluronic F68 adsorbs as slightly elongated structures which are
about 1.5 nm in height, 42 nm wide and 70 nm long. Moreover, similar to PVP preferential
adsorption can be detected along the crystal’s atomic steps. Multiple scans of the same area
resulted in migration of the adsorbed Pluronic F127. Similar multiple scans with all other
polymers did not reveal any tendency for migration or dislocation of the adsorbed polymer. HPC
adsorbed on the ibuprofen surface in an extended open chain pattern similar to HPMC. The
observed heights and widths were also comparable to those observed for HPMC approximately 3

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and 40 nm, respectively. Furthermore, significant differences in the adhesion forces between the
bare surface and the regions with adsorbed polymer were clearly revealed for PVP. A minor
difference in for HPMC and the bare ibuprofen surface was apparent, though barely statistically
significant. Therefore, it was evidenced that the HPMC was the best polymer among those

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investigated in terms of interaction with the ibuprofen surface to provide an effective surface

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coverage.
HPMC is a substituted cellulosic polymer with methoxy and hydroxypropyl substitution at

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the 1, 3, or 6 positions of the repeating anhydroglucose units [72, 73]. The authors hypothesized

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that interactions between the hydrophobic backbone of HPMC and the hydrophobic groups
present on the ibuprofen surface were responsible for its extensive and strong adsorption. High

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affinity of the HPMC for the ibuprofen surface also caused the molecules to be adsorbed in an
open-chain-like pattern as compared to a compact/ coiled structure.
By assistance of the AFM, direct visualization of the adsorbed stabilizer directly on the drug
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surface would be aid in comprehending the extent of interaction between the drug and the
stabilizer and providing vital information about the characteristics of the adsorbed layer such as
the specific arrangement of the stabilizer molecules, the thickness of the adsorbed layer, and the
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strength of the interfacial film, which is significant for predicting the wettability, aggregation,
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crystal growth and Ostwald ripening of the prepared nanosuspensions. Therefore, AFM method
could be employed for screening and predicting the most suitable stabilizers during the
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nanosuspensions preparing.
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3.4. The hydrophobic moieties of the stabilizers

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For a stable nanosuspensions preparing, the hydrophobic moieties of the selected stabilizers
are very important. Firstly, the force of driving the stabilizers adsorb onto the surface of
hydrophobic drug is the hydrophobic moieties in the stabilizers; thereby the surface anchored
stabilizers provide steric or ionic stabilization. Without the adsorption, stabilization cannot occur,
and so no dispersed nanosuspensions resulting from any preparing approaches [74]. Secondly, the
stabilizers should have high hydrophilic property. Because of its hydrophilic nature, the segment
will tend to locate itself toward water instead of hydrophobic drug surface. Thus, it can provide
effective steric or ionic stabilization for drug nanosuspensions. However, like the conventional
surfactants of small molecular weights, it may excessively help the dissolution of hydrophobic
drugs, resulting in the destruction of crystalline structures. The destruction produces micelle like
structures in liquid formulations. The suitable hydrophobic moieties of the stabilizers for
completed stabilization the mechanical fractured or nano-precipitated nanocrystals by the nearby
stabilizers in a short time period is very important

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It was reported that not the morphology but the hydrophobicity of stabilizers was the most
critical parameter in the preparation of drug nanosuspensions. And the effective stabilization
performance of the stabilizers requires the hydrophobic moiety content to be higher than 15 mol%
[18]. To compare effects of hydrophobicity on nanosuspensions processing, random and block

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copolymers based on lysine (K), phenylalanine (F), leucine (L), and alanine (A), which has quite

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different hydrophobicities, were prepared via ring opening polymerization and were used for
naproxen nanosuspensions preparation. While the hydrophilic moiety of copolymers, K used as

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the hydrophilic moiety of copolymers, three other amino acids, F, L, and A, were used as

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hydrophobic moieties. The use of different amino acids imparts a different hydrophobicity to
amino acid copolymers. The molecular weight of the amphiphilic amino acid copolymers was

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5000-25,000 g/mol. If all other factors such as the K content, polymer morphology, etc., remain
the same, the hydrophobicity of copolymers was the net changes in the driving force of the
absorption of the copolymers onto hydrophobic drug surfaces. Desorption was also related with
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the hydrophobicity of copolymers. As the content of hydrophobic moieties increases, the
adsorption of copolymers was enhanced. When the hydrophobic content is above 15 mol%, the
mean particle size can be reduced to below 400 nm. Below 15 mol%, it spans a wide size range of
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500-1500 nm. Above 15 mol%, a significant change in mean particle sizes was not observed.
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Therefore, for successful polymer adsorption and particle size reduction, the mole fraction of the
hydrophobic moieties needed to be at least 15 mol%. Furthermore, the morphology of copolymers
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was not an important factor in determining particle size reduction. Once wet comminution in the
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presence of a proper amino acid copolymer produced drug nanosuspensions, their particle size
were found to be stable up to 30 days without significant aggregation.
Beside the above mentioned typical stabilizers of amphiphilic amino acid copolymers, the
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hydrophobic moieties also played a key role in achieving stable nanosuspensions in the proteins
type stabilizers. Increasing the content of hydrophobic moieties, the adsorption of proteins might
be enhanced to some extent. In comparison with other common stabilizers like HPMC, PVPK30
and PEG 6000, the denatured whey protein isolate (WPI), soybean protein isolate (SPI) and
β-lactoglobulin (β-lg) had much more hydrophobic residues (amino acids and disulfide bonds)
[70]. Thus, the denatured proteins could provide higher affinity with the drug surface and better
stabilization effect.

3.5. The optimized concentration and temperature for the given stabilizers

The concentration of stabilizers is of the foremost importance for the stabilization of

nanosuspensions, and as a result, the amount of stabilizer added in the formulation should be
optimized. The concentration of surfactant type stabilizers should be used under the critical

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micelle concentration (CMC). However, the usage of an inadequate amount of stabilizer will not
provide a complete coverage of the drug molecule surface which is needed to provide steric
repulsion between the nanoparticles in suspension [16]. Conversely, the concentration above the
CMC, micelles will be formed. The formation of the micelles plays a critical role in thermal

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instability of the prepared nanosuspensions. After the attainment of the CMC, the micelles began

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to compete for surface adsorption so that the total adsorption at the interface begins to decrease as
the micelles become more numerous. Therefore, concentration higher than CMC could actually

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mean less surfactant adsorption, which would further destabilize the nanosuspensions and thereby

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contributed to particle size increasing [75, 76].
Furthermore, at high stabilizer concentrations, much above of the plateau of the adsorption

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isotherm, electrostatic stabilizers could cause a decrease in the diffuse layer, which leading to a
decreased zeta potential and thereby decreased physical stability [58]. Furthermore, for the
polymer concentration in the nanosuspensions dispersion, it is usually stabilized by increasing to
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the order of 1%.
For the paclitaxel nanocrystals preparing, Pluronic F127 was selected as stabilizer [77]. In the
preparing, attempt to further stabilize the nanosuspensions against thermal induced aggregation,
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more Pluronic F127 surfactant was added in the formulation. However, it was resulted in
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formation of micelles that worsened the stability of nanosuspensions. The F127 desorption
experiment suggested different surfactant adsorption affinity to nanosuspensions surface below
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and above the CMC. Below the CMC monomers bound to nanosuspensions surface with high
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affinity, but above the CMC low affinity surfactant aggregates readily left the surface upon
dilution. The reason for why more F127 worsened the stability of nanocrystals could be revealed
in the Fig. 4. It could be observed by the TEM that instead of forming a thicker layer on the
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nanocrystals surface for better stabilization, micelles began to form in solution at higher
concentration of F127 surfactant. The CMC of Pluronic F127 at 25 °C is 7.19×10-5 M. The
concentration of F127 in nanocrystals prepared with 1:5 (w/w) of paclitaxel/F127 is below the
CMC of F127. However, for 1:10, 1:20, and 1:30 of paclitaxel /F127, the concentration of
surfactant goes beyond the CMC. It was reported that a suitable working polymer to drug ratio
(steric stabilizer) is from 0.05:1 to 0.5:1 [78].
Beside the influence of stabilizers concentration, the temperature of is also an important
factor for determining the stability of the prepared nanosuspensions. The hydrophobic interaction
is the mostly manner between the nanosuspensions and stabilizers and this is a negative entropic
processes. Thereby, the higher the temperature of the nanosuspensions, the more
thermodynamically unfavorable the system stability becomes. So, in hydrophobic
nanosuspensions system, the tendency of aggregation is enhanced at higher temperature [77]. This

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predication was confirmed by the paclitaxel nanosuspensions. The particle size of fresh prepared
paclitaxel nanosuspensions was about 176 nm. However, after incubation at 37 °C for 2 h, longer
crystals were growth and the particle size increased to 258 nm [77]. At higher temperatures the
surfactant adsorption is inhibited more than micellization, which indicated that micellization will

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out-compete surface adsorption. Thereby, monomers on the surface of nanosuspensions started to

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aggregate at higher temperature and were drawn to leave the crystal surface upon dilution, making
the crystal prone to aggregate.

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Furthermore, according to the Stokes-Einstein equation, an increasing in temperature will be

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resulted in a decreasing in dynamic viscosity and an increasing in the diffusion constant [79]. A
higher diffusion constant leads to a faster diffusion of the nanosuspensions. Possessing a higher

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kinetic energy, the electrostatic repulsion between the particles could be easily overcome which
also will resulted in the nanosuspensions aggregation. For example, quercetin nanosuspension
stored at 40 °C was more instable than the stored at 25 °C and 4 °C [80]. Similarly result was also
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revealed on other six nanosuspensions formulations [71].
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Fig. 4. TEM pictures of paclitaxel nanocrystals revealed the effects of stabilizer concentration and
temperature for the nanocrystals thermal stability. (A) paclitaxel/F127 (1:10, w/w) and (B)
paclitaxel/F127 (1:20, w/w), the white spherical structures in the background are micelles formed
at high concentration of F127. (C) Nanocrystals before 37 °C incubation. Measured size: 176 nm
(D) longer crystals formed by incubation at 37 °C for 2 h. Measured size: 258 nm (adopted from
[77]).

3.6. The pH environments for optimizing stabilizers functions

The aqueous media pH plays an important role in the influencing of the stabilizers function
on the stabilizing nanosuspensions. The influences of pH on the stabilizers functions were
demonstrated in food proteins formulated indomethacin nanosuspensions [70]. The isoelectric
points (pI) of the three denatured food proteins, whey protein isolate (WPI), soybean protein

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isolate (SPI) and β-lactoglobulin (β-lg), falls in the range of 4-6. At the point of pI, there is limited
stabilization effect on the nanosuspensions because the charge of the proteins is zero. This leads to
particle aggregation. At pHs above or below the pI, the food proteins indicate amphiphilic
properties and can work as stabilizers. However, at acidic pH (<3.5) or highly basic pH, food

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proteins may dissociate into monomers, which compromises their stabilization efficiency. Slightly

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basic pHs seems to be beneficial to food protein stabilizers. Similarly results also revealed on
dipropionate nanosuspensions using another kind of biopolymer (class II hydrophobin) as

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stabilizers which indicated that superior nanosuspensions stabilization effect was observed by

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more than 2.5- fold at pH 8 [81].

3.7. The drugs should have little solubility in the stabilizers solution

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The solubility of the formulated drug in the stabilizer solution plays a significant role in the
increasing in particle size on storage, indicating Ostwald ripening. As a stabilizer of the
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formulated nanosuspensions, it should be possess little effects on the drug solubility. Otherwise, it
may be induced the agglomeration and/or Ostwald ripening of the nanosuspensions colloid system.
For example, denatured food proteins whey protein isolate (WPI), soybean protein isolate (SPI)
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and β-lactoglobulin (β-lg) formulated indomethacin nanosuspensions displayed highlighting

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superior stabilization and significantly smaller than that observed for common stabilizers prepared
nanosuspensions. Compared with the purified lecithin, Tween® 80 and Pluronic F68
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nanosuspensions, this result could be attributed to the effect of Ostwald ripening due to relatively
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higher indomethacin solubility in presence of the stabilizers, which led to agglomeration and
crystal growth of the drug particles. In fact, many other reports were also indicated that an
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increasing in intrinsic solubility of the drug by stabilizers like sodium dodecyl sulfate, Tween® 80
and Pluronic, would lead to Ostwald ripening and agglomeration [70].
The similarly results was also reported by Verma et al [82]. In that study, two ibuprofen
nanosuspensions, stabilized by various stabilizers (SLS, PVP K30, Pluronic F68 and F127,
Tween® 80 and HPMC), was prepared by milling via microfluidization or precipitation via
sonication. Both the processes gave similar initial formulations with comparable short-term
stability. In the microfluidization process, the drug solubility in the stabilizer solutions plays a
dominant role. There was a relationship between drug solubility in the stabilizer solution and
stability of the particle size observed on storage. The stabilizers (PVP K30, Pluronic F68 and
HPMC) which minimally affect the intrinsic aqueous solubility of the ibuprofen (0.049 mg/ml)
resulted in lower mean particle size compared to stabilizers that significantly increased ibuprofen
solubility (SLS, Tween® 80 and Pluronic F127). Higher ibuprofen solubility during processing and
storage in the presence of SLS, Tween® 80 and Pluronic F127 can lead to Ostwald ripening.

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Therefore, only stabilizers which have a minimal/negligible effect on drug solubility should be
used in the preparation of nanosuspensions.
Beside the agglomeration and/or Ostwald ripening, a drug is soluble in the stabilizer in the
solid state also could induced an amorphous solid dispersion formed at the interface of the

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formulated nanosuspensions [4].

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3.8. The influence of stabilizers on morphological structure of the nanocrystals

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The stabilizers are playing a critical role in the nanosuspensions solidification. Firstly, it

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should be able to achieve controlled crystallization during stabilization and secondly to increase
the wettability of hydrophobic drug in dissolution medium [45]. Among all the effect factors, the

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morphological structure of the solidified nanosuspensions is a critical parameter for determining
the wettability and re-dispersibility of the prepared products. Beside of the two aspects, the
different stabilizers resulted in the different morphology also is a potential factor for influencing
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the stability of the nanosuspensions [83]. For example, the stability between the two
nanosuspensions shaped as foliated and sphered may be different with each other. The main reason
for the different stability is the different dispersibility and surface area between the two shapes
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[84]. Generally, the morphology of the solidified nanosuspensions could be revealed by SEM
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technique.
SEM images revealed distinct differences in the morphological structure of celecoxib
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nanosuspensions influenced by the stabilizers (Fig. 5) [12]. Celecoxib is a selective COX-2

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inhibitor with low water solubility. The celecoxib nanosuspensions were prepared by the
emulsion-diffusion method using three different stabilizers (Tween® 80, PVP K30 and SDS).
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Spray-dried nanosuspension was blended with microcrystalline cellulose and obtained solidified
nanocrystals. The nanosuspensions stabilized with PVP K30 and SDS showed advantages over
Tween® 80 due to sticking of the dried product and unexpected changes observed on DSC curves.
The raw micronized celecoxib API was predominantly needle-shaped crystals with sizes between
2 and 100 μm. The SEM images of Tween® 80 stabilized nanosuspensions showed some particles
with size below 200 nm together with larger plates. The reason for this phenomenon is that the
Tween® 80 is liquid in the room temperature. Therefore, when the Tween® 80 stabilized
nanosuspensions drying, the nanocrystals may be adhered and aggregated with each other.
Meanwhile, PVP/SDS stabilized nanosuspensions observed as thin plates aggregated into spheres.
And all images revealed nano-sized particles in at least one dimension but almost all were very
thin, estimated to 50 nm. This morphology changes induced by the stabilizers may be a potential
influence factor for the nanosuspensions stability. Furthermore, it could be generalized from this
work that liquid stabilizers prepared nanosuspensions not suitable for solidification process.

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Fig. 5. SEM images of the celecoxib nanocrystals with different stabilizers following spray-drying

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confirmed the influence of stabilizers on the morphological structure of the nanocrystals. (a)
celecoxib API (size bar 10 μm); (b) Tween® 80 nanosuspension (size bar 1 μm); (c and d)
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spray-dried nanosuspension stabilized with PVP/SDS (size bars 300, 200 nm respectively)
(adopted from [12]).

3.9. The influence of stabilizers on nanocrystals particle size

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The types of non-ionic and ionic polyelectrolytes surfactant are the commonly used
stabilizers for the nanosuspensions formulation. The CMC and hydrophile-lipophile balance (HLB)
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of each surfactant are different with others. Therefore, the selected stabilizers may be influencing
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the size and stability of the prepared nanosuspensions. Furthermore, the combination of the
different type of stabilizers may be further enhancing the influencing. As entropic steric
interactions are more sensitive to thermal fluctuations than electrostatic repulsions, it’s a common
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usage of the combination of non-ionic and ionic polyelectrolytes. Non-ionic surface modifiers are
known to form a steric mechanical layer while ionic surface modifiers form electrostatic repulsive
layer to prevent aggregation of particles in the process of size reduction.
Pyrimethamine nanosuspensions, prepared by combine of nanoprecipitation and high
pressure homogenization technique, evaluated the effective of combination of non-ionic Pluronic
F68 with either anionic gellan gum (GG) or cationic PEI on particle size reduction using factorial
design approach [62]. It was revealed that the anionic stabilizer had more pronounced effect on
size reduction compared to non-ionic F68. When the concentration of GG increased from 0.05%
to 0.1% (w/v), an increasing in particle size was observed. Meanwhile, if the concentration of GG
higher than 0.1% (w/v), the particle size of the nanosuspensions decreased. The combined effect
of non-ionic stabilizer F68 and cationic stabilizer PEI was revealed that the concentrations of both
variables showed a pronounced effect on particle size reduction. As the concentration of either
stabilizers increased, particle size significantly reduced.

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Rachmawati and coworkers investigated the influence of five stabilizers on the particle size
of curcumin nanosuspensions [24]. Curcumin is a nature product of polyphenolic phytoconstituent
and water insoluble under acidic or neutral conditions and dissolves in alkaline environment but
unstable undergoing rapid hydrolytic degradation in neutral or alkaline conditions. It was

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formulated as nanosuspensions by HPH approach under different stabilizers. After 20

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homogenization cycles, smallest size was obtained for the TPGS stabilized system, the size
increased in the order of: TPGS < SDS < PVP < PVA < Na-CMC. The HLB of TPGS is about of

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13. The TPGS has bulk hydrophilic tail (polyethylene glycol) and hydrophobic portion (tocopherol

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succinate) as well as large surface areas. The combination of low viscosity and high surface
activity makes it as a good stabilizer for nanosuspensions stabilities and superior to other

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stabilizers used in the production. Furthermore, this ordering also revealed that the lower the
molecular weight of surfactants, the faster they diffuse and vice versa.
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3.10. The influence of stabilizers on zeta potential

Zeta potential is the potential at the hydrodynamic shear plane and can be determined from
particle mobility under an electric field. The mobility will depend on surface charge and
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electrolyte concentration of employed stabilizers. As the zeta potential is one of the factors
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determining the physical stability of dispersion system, its value can be used to predict the
physical stability of the prepared nanosuspensions [24]. Generally, for a physically stable
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nanosuspensions solely stabilized by electrostatic repulsion, a zeta potential of ± 30mV is required

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as a minimum [85]. Combined with the steric stabilization, the absolute value of zeta potential
about 20 mV is sufficient to fully stabilize the nanosuspensions system. The commonly used
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electrostatic stabilizer includes Tween® 80, SLS, SDS and docusate sodium (DOSS). For a certain
candidate compounds, how to select a suitable zeta potential stabilizers and which zeta potential
high enough for the stability is a puzzle. Furthermore, to investigate the influence of the other
stabilizers on the zeta potential changes also is an important for the nanosuspensions formulating.
Zeta potential of glyburide nanosuspensions was found to dependent more on the polymer
concentration compared with surfactant concentration in the formulation stabilized by HPMC and
SLS [58]. HPMC is a nonionic polymer and SLS is an anionic surfactant. The zeta potential values
increased at low level of ratio of polymer to drug where the level of ratio of surfactant to drug is
high. At low level of ratio of polymer to drug, the particle surface of drug was not covered so
densely with polymer, due to which anionic surfactant had more access to the surface of drug if
the concentration of polymer was lower. Adsorption of anionic surfactant onto the particle surface
leading to high zeta potential value, such as ibuprofen nanosuspensions stabilized by SLS [82].
The zeta potential value of the nanosuspensions decreases at high level of ratio of polymer to drug,

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irrespective of ratio of surfactant to drug. At this stage due to increased concentration of polymer
in nanosuspensions its adsorption on drug particles increases which leads to a less electrostatic
interaction with anionic surfactant than if the negatively charged glyburide surface was exposed,
this was why the zeta potential goes down.

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In a similar study, meloxicam nanosuspensions formulated with the same stabilizers as the

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glyburide nanosuspensions, HPMC and SLS, was prepared by the same approach, media milling
[41]. The influence of HPMC and SLS on the zeta potential of the meloxicam nanosuspensions

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revealed the similar results to the glyburide nanosuspensions [58]. Thus, the zeta potential of the

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nanosuspensions was depended more on the polymer concentration compared to surfactant
concentration.

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Although the application of high concentration polymers as stabilizers may be reduce the zeta
potential of the prepared nanosuspensions, it is often the case that the polymers bridge the
particles and the dispersion is destabilized, if the polymers concentration is very low [24]. In case
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of polymers used in the preparation of nanosuspensions, these molecules should adsorb onto the
surfaces of drug nanocrystals and provide steric stabilization effect. Therefore, it should to seek a
balance and cross point between the zeta potential provided electrostatic repulsion stabilization
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and the polymers provide steric stabilization.

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4. The influences of preparation approach on stability

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4.1. High pressure homogenization approach

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The technologies of nanosuspensions preparation have significantly advanced over the last
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several decades. Milling, high pressure homogenization (HPH), impinging jet, electro-spraying,
liquid-based methods, and supercritical fluid processes are the technologies that are currently
available or being actively developed for nanosuspensions preparation [18]. Furthermore, the
bottom-up approach, such as nano- precipitation, also is an effectively technique for the preparing
[86]. And the combination of the above mentioned methods extensive the techniques for the
nanosuspensions preparing. For the preparation methods, it has been extensively discussed in
previously research and review articles. Accompanying with particle size reduction, energy inputs
or surface stabilization compensate the dramatic increase in surface energy (extra Gibbs free
energy) are needed. Thus, all the approaches for the nanosuspensions are of thermodynamic
instability and kinetic instability processing. However, the different methods involve the different
theories in particle size decreasing and surface area increasing. Therefore, the potential influence
the different technique for nanosuspensions preparing on the stability is a critical factor for the
preparation technique selection.

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Generally, the nanosuspensions morphology and stability is influenced by manufacture

approach and operating parameters. For example, the HPH process may be obtained short needle
shaped nanosuspensions and during the storage, the length of the needle may be increased by
Ostwald ripening. Furthermore, the HPH operating not induced the crystal form transformation.

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Compared with the HPH approach, the media milling sometimes could significantly damage the

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crystallinity of particles and resulted in sphericity nanocrystals. Similarly with the media milling
approach, the bottom-up technique also could be induced crystal form transformation, but resulted

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in needle shaped nanosuspensions. Furthermore, the bottom-up method prepared was more liable

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to be Ostwald ripening and particle size increasing.
HPH approach relies on the forcing of a suspension through a small gap which makes

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miniaturization of this technology less straightforward [87]. Compared with the medium mill
approach, the potential advantages of the HPH technology for nanosuspensions preparation
include saving time, avoidance of contamination with erosion from milling pearls and
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microbiological problems [21]. It was reported that the HPH prepared nanosuspensions could to
protect chemical labile drug from degradation [28]. Omeprazole is a substituted benzimidazole
that selectively inhibits the gastric proton pump of the parietal cells. However, the currently
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available formulations should be used within 6 h due to the chemical instability. One approach for
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the stabilization of chemically labile molecules in solution is to transform the soluble molecules
into solid nanosuspensions. Therefore, Möschwitzer and coworker investigated the possibility of
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protecting omperazole from degradation by using the HPH technique. Compared with the solution
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formulation stored at the same conditions, the omeprazole nanosuspensions demonstrated an

excellent stability and the nanosuspensions showed no significant alteration in drug content and in
color over the monitored period. Meanwhile, a significant loss of omeprazole and discoloration
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was identifiable in the solution. Thus, the HPH prepared drug nanosuspensions showed excellent
chemical stability compared to drug solutions. And this technique is an alternatively suitable
approach for preparing drug nanosuspensions formulations to protect chemical labile drugs from
degradation.
During the HPH operation, homogenization pressure is the main parameters which
determining the final particle size and crystal form of the prepared nanosuspensions [88]. SN
30191 is an inhibitor of phosphatidylinositol 3-kinase (PI3-K) and an anti-cancer candidate
compound synthesized by the Auckland Cancer Society Research Centre (ACSRC), the University
of Auckland, New Zealand. It was found to exist in two polymorphic forms (I and II) and a
hydrate with an equilibrium solubility < 0.1 μg/ml (pH 1.3-11.0, 37 °C). SN 30191 was
formulated as nanosuspensions through HPH approach and revealed the crystallinity
transformation [89]. During the homogenization process, solid state transformations were

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observed in forms I and II as a function of pressure. A further increase in pressure resulted in

formation of a hydrate. This transformation was demonstrated by powder x-ray diffraction (PXRD)
patterns SEM micrographs (Fig. 6). PXRD peaks were intense and sharp for form I compared to
form II, indicating that form II probably has a more disordered structure. Moreover, at high 2θ

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angles (20-25°), form I peaks were more intense than those for form II, indicating a high degree of

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crystallinity for form I. The PXRD pattern of the hydrate was different from those of forms I and
II. This showed that all the three solid forms have different molecular arrangements in their crystal

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structure. As revealed by SEM, Form I comprised irregular shaped crystals with a non-uniform

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surface, whereas form II crystals showed a fibrous appearance. The hydrate showed the presence
of a mix of thin needle shaped crystals and fibers. In the nanosuspensions homogenization, low

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pressure mechanical activation (as applied by ultra-turrax) led to the transformation of form II-I,
though this transformation was relatively slow. When high pressure mechanical activation was
applied using a high pressure homogenizer, transformation of form II to form I was rapid.
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Furthermore, hydrate formation was observed only after mechanical activation of form I at high
pressure. Therefore, the transformation of form II-I and then to hydrate involves a considerable
energy barrier which was overcome by application of mechanical energy.
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Fig. 6. PXRD and SEM micrographs revealed the influences of HPH pressure on the SN 30191
crystallization. A, Form Ⅰ , at low homogenization pressure; B, Form Ⅱ , the initial
crystallization; C, Hydrate (adopted from [89]).
Beside the influence of homogenization pressure on the crystalline, the combination of
freeze-drying with HPH also influences the particle size reduction effectiveness of the
homogenization [90]. The model drug glibenclamide was dissolved in mixtures of dimethyl
sulfoxide (DMSO): tert-butanol (TBA) (10%/90%, v/v), freeze-dried and subsequently

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homogenized at high pressure. Under the same homogenization conditions after 20 cycles, the
particle size of freeze-dried glibenclamide prepared nanosuspensions was 164 nm (z-average) and
0.114 μm (d50%). On the contrary, the particle size untreated glibenclamide prepared
nanosuspensions was 772 nm (z-average) and 2.686 μm (d50%). This significance in particle size

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was a potential factor for resulting in different stability of the two nanosuspensions as there was

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existing different Gibbs free energy between the two nanosuspensions systems.

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4.2. Media milling approach

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Among the different techniques used for the preparation of nanocrystals, wet media milling is
considered to be an attractive approach which would allow for a relatively easy scale-up with

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respect to nanosuspensions pharmaceutics industrial production [58]. Media milling could be
performed by agitation of devices containing the starting suspension and milling media. The drug
particle size reduction during the wet media milling seems to be a complex process, where
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multiple factors need to be considered in order to produce optimal nanosuspensions. During the
milling process, the drug crystals break into smaller particles and thus generate fresh surfaces
continuously [16]. Therefore, the time gap between the rate of new surface generation and the time
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of stabilizers absorbed onto the new surface determined the stability and the efficiency of the
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nanosuspensions and the wet milling.

The media milling process of drug nanosuspensions in the presence of stabilizers is an
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effective method for the nanosuspensions preparing. The key of drug nanosuspensions processing
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is how to compensate the extra free energy of freshly exposed surfaces. In media milling process,
the degree of compensation depends on the interactions between drugs and stabilizers [65].
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Furthermore, retaining the crystallinity of drug particles during processing is beneficial to the
stability of drugs [57]. However, media milling sometimes could significantly damage the
crystallinity of particles. Therefore, the biggest concern in the media milling is the shear induced
amorphous drug formation. Compared to its crystalline form, amorphous drug form is
characterized with enhanced solubility and Ostwald ripening [71]. Caution needs to be taken in the
generalization of pharmaceutical active ingredients, since each drug crystalline form has different
physical stability [18].
The crystal defects and re-crystallization of amorphous is a potential instability of the
nanosuspensions during preparation and storage. In the media milling processes considerable heat
is generated which may cause degradation of heat sensitive APIs. The media milling approach also
may cause mechanical activation at drug particle surfaces. Crystal defects due to disordering of
the crystal surface and generation of localized amorphous regions have been implicated in
increased surface energetic. Reordering of crystal defects and re-crystallization of amorphous

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regions has resulted in both physical and chemical instability of processed materials on storage
[82]. Joshi et al has been observed an increase in the specific surface area of budesonide on
storage after the milling process [91].

4.3. Microfluidizer approach

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The microfluidizer technology was patented in 1985 and was first reported in a scientific
publication in 1987 [92]. In microfluidizer, the crude liquid suspensions is pumped and

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accelerated with a high speed to an interaction chamber. In the “Z” type chamber, the suspension

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changes directions of its flow leading to shear forces and particle collision. And in the “Y” type
chamber, the suspension stream is divided into two micro-streams which then collide against each

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other and produce exceptionally fine nanosuspensions [86].
Compared with the commonly used HPH approach, the microfluidizer technology is more
effectively and saving time. Furthermore, it is reported that the use of a microfluidizer is preferred
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for the milling of drug crystals which are of comparatively lower hardness than crystals used in
wet media milling [16]. Currently, some water insoluble compounds, such as bifendate and
amoitone B, have been developed as nanosuspensions by microfluidization method [93, 94].
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Compared with the evaporative precipitation method, Liu et al prepared Riccardin D

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nanosuspensions by microfluidization approach [86]. It was found that the two processes were
simple and adequate to make nanosuspensions, and the products were very stable.
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The influence of three commonly used nanosuspensions preparing approaches, HPH, media
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milling and cavi-precipitation, on the stability of the produced quercetin nanosuspensions were
investigated by Mitali et al [80]. For the long-term physical stability study, the three method
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prepared nanosuspensions were stored at three temperatures (4 °C, 25 °C and 40 °C) over a period
of 180 days. The nanosuspensions prepared by HPH technique were physically stable over the
investigated period at 4 °C and 25 °C. The light microscopic pictures confirmed that almost all the
quercetin nanosuspensions were finely dispersed and there were hardly any agglomerations.
Nevertheless, the quercetin nanosuspensions, prepared by media milling, were observed slight
aggregations after 180 days of storage at 25 °C. The cavi-precipitated method prepared quercetin
nanosuspensions, with DMSO and ethanol solvent, were the most instable formulation over a
period of 180 days. Due to agglomeration and Ostwald ripening, LD95 of the particle distribution
was more than 1 mm at 90 days and just doubled after 180 days. As observed from the SEM
images (Fig. 7), the high-pressure homogenized and media-milled quercetin nanosuspensions
showed better stability than the cavi-precipitated nanosuspensions in 180 days. The
cavi-precipitated nanosuspensions showed the poorest stability due to aggregation and
agglomerations judging from the particle size determination and the SEM image. Besides the

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Ostwald ripening phenomenon, recrystallization and agglomeration, the solvents used during
cavi-precipitation might have competed with the surfactant for hydration leading to a partial
dehydration of the surfactant, which subsequently affected the stability of the quercetin
nanosuspensions.

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Fig. 7. SEM images of original quercetin API powder (A; bar = 10 mm) and quercetin
nanosuspensions (bar = 1 mm) after 180 days of storage at room temperature (25 °C). B: 5% w/w
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quercetin nanosuspensions produced by HPH; C: 5% w/w quercetin nanosuspensions produced by

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bead milling; D: cavi-precipitated nanosuspensions with ethanol (adopted from [80]).

4.4. Bottom-up approach

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Compared with the potential instable factors of the top-down approach, the bottom-up
technique may adversely influencing nanosuspensions formulations, such as the generation of
various unstable polymorphs, hydrates and solvates during processing [82]. Furthermore, the
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bottom-up approach involved the solvents usage which is usually difficult to completely remove
[95, 96]. Therefore, a proper control of residual solvent levels is required as a consequence of the
potential toxicity of organic solvents. And the usage of organic solvents is also related to a number
of challenges as proper and safe handling of flammable and/or explosive solvents which requires
special facilities and equipment [90]. Furthermore, any residual solvent can cause physical and
chemical instability of the formulation. Moreover, the bottom-up approach usually resulted in
needle shaped particles due to the rapid growth in one direction which influences the physical
stability of the nanosuspensions [40].

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5. The solidification methods for improving nanosuspensions stability

5.1. Spray drying

Compared with the nanosuspensions state in the aqueous medium, the solidified state is more

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preferred, as the aggregation and other instable factors are significantly decreased [33]. Therefore,

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the prepared nanosuspensions commonly converted into solid state by certain approach. And then

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the solidified powder was formulated as other dosage forms, such as sterile powder for injection,
nebulized for pulmonary delivery, tablets and capsules for oral delivery [12, 97-99]. The end

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dosage forms of the nanosuspensions avoided the aggregation, hydrolysis and other instable
representation [35]. During the process, the solidification operation is the critical point for all the

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final products.
Generally, the particle aggregation during the solidification process was inevitable. In
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aqueous conditions, the surface energy of particles is counterbalanced by steric or ionic
stabilization [18]. However, in the solidified states, stabilizers chains solidify themselves and their
stabilization can be no longer effective, resulting in significant aggregation as could be found in
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the previous study [100]. Therefore, to select a suitable method for nanosuspensions solidification
is key point in nanosuspensions preparing [101].
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Own to less time and energy consuming, spray drying is generally preferred over
freeze-drying by pharmaceutical industry on transform liquid nanosuspensions into a dry product
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[101, 102]. Celecoxib nanosuspensions prepared with different stabilizers followed spray drying
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[12]. It was found that drying of the nanosuspensions stabilized with Tween® 80 was problematic
due to the sticking of the product to the wall of the drying chamber. Fortunately, this phenomenon
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was not observed when the nanosuspensions stabilized with PVP/SDS.

Similar to the spray drying, electro-spray drying technique also could be used as a novel
strategy in the preparation of redispersible drug nanosuspensions formulations without
aggregation [103]. In the equipment, electrohydrodynamic force is instead of the pressurized air
stream for atomization [104]. The applied electrical potential breaks up the droplets with an
accelerating drying process, while the rapid early stage of the drying procedure seems to prevent
irreversible aggregation between drug nanocrystals [105]. Thereby, this method could used to
maintain the dispersion of the nanosuspensions before and following solidification [106]. At 2.7
kV, redispersible dried naproxen nanocrystals were obtained from drug nanosuspensions prepared
via wet comminution (Fig. 8) [103]. According to the theoretical analysis, the droplets prepared
with an electrical potential of 2.7 kV should be quite small and thus, fast drying is expected.
Therefore, enough consolidation could occur before HPC chains become entangled or drug
particles fuse together. If enough drying precedes the contact between drug nanosuspensions, there

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will be no chance of aggregation. Furthermore, a dry HPC layer on the surface of the drug
nanosuspensions would serve as a protective layer and allow for excellent redispersibility.

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Fig. 8. SEM micrographs of particles prepared by electro-spray drying at various voltages
(naproxen nanosuspension concentration = 8 wt%). (a) 0 kV, (b) 1.0 kV, (c) 2.0 kV, and (d) 2.7 kV.
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The higher voltage, the less aggregation of the solidified nanosuspensions was observed (adopted
from [103]).
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5.2. Freeze-drying
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Technically, nanosuspensions solidification process also could be achieved using established

other unit-operations such as freeze-drying [57]. For thermo-labile drugs, such as protein drugs
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and vaccines, freeze-drying is the most suitable approach for the solidification process [107]. And
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freeze-drying is also a common process to retain the redispersibility of drug nanoparticulates after
solidification. Freeze-drying removes water from a frozen sample through sublimation. The first
step is freezing, followed by primary drying (ice sublimation) and secondary drying (desorption of
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unfrozen water). The previous studies presented a comprehensive understanding of the influence
of various parameters on the freeze-drying process and promoted the application of the
freeze-drying technique in nanosuspensions drying [99, 108]. Among these parameters, the type of
cryoprotectant and the freezing rate were two critical factors among the whole process.
In general, a cryoprotectant is usually added to the fresh prepared nanosuspensions to avoid
freeze damage due to ice formation and to avoid particles aggregation [109]. The choice of the
cryoprotectant is a critical point, as it needs to be ensured that the nanosuspensions powder can be
re-dispersed as separated particles and don’t aggregate, otherwise would lead to a loss of their
special properties [110]. Theoretically, during the freeze-drying the cryoprotectant could be
replaced the function of water and maintained the dispersion state of the aqueous nanosuspensions
systems. When a cryoprotectant is present, water freezes into ice crystals while excluding solutes
and particles into a cryoconcentrated liquid phase. Furthermore, the cryoprotectants can replace

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water and/or other liquid molecules, with a newly formed strong layer that combines closely with
nanosuspensions during the sublimation process of the freeze-drying process. Thus, the newly
formed layer effectively prevents aggregation and ripening of the nanosuspensions due to the
chemical and structural properties of the cryoprotectants. Usually, multi-hydroxy compounds

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served as cryoprotectants. The multi-hydroxy compounds can maintain the spatial orientation and

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distance among the nanosuspensions, when the ice sublimes during the freeze-drying process. In
consequence, the nano-dispersion systems are prevented from forming the aggregates [108].

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Currently, a series of polymer cryoprotectants, such as polyatomic alcohols, were usually

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selected to stabilize the nanosuspensions during the freeze-drying process. The most used
polyatomic alcohols including mannitol, lactose, sucrose, trehalose, glucose, et al. The above

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mentioned cryoprotectants, highly water-soluble agents, could procure the ability of creating a
highly hydrophilic environment around nanosuspensions. In the Deacety Mycoepoxydiene
nanosuspensions formulations, mannitol was the most successful agent of all the evaluated
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cryoprotectants, and the nanosuspensions solidified with mannitol (5 wt.%) was chosen for further
investigations [83]. Beside the type of cryoprotectants used for the freeze-drying, the amount of
the selected cryoprotectants also could be to influence the stability of the solidified
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nanosuspensions. It is generally known that a higher concentration of cryoprotectant and faster

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freezing result in better nanosuspensions redispersibility. However, higher amounts of sucrose

unexpectedly resulted in increasing agglomerate after itraconazole nanosuspensions freeze-drying
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operating [59]. Before the freeze-drying, the sucrose (50,100 and 200%, relative to the weight of
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itraconazole) was added as a cryoprotectants. It was revealed that containing higher amounts of
the sucrose, larger agglomerates were observed [laser diffractometry average diameters: 69.8 ± 7.7
μm (0%), 55.4 ± 26.7 μm (50%), 128.2 ± 7.8 μm (100%) and 219.1 ± 154.1 μm (200%)].
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Therefore, with an increasing in the cryoprotectant concentration, the irreversible aggregation of

nanosuspensions may either increasing or decreasing depended on the system of the
nanosuspensions and the freezedrying conditions.
Furthermore, the speed of freezing rate, determined mainly by temperature gradient, is also a
critical parameter influencing on the stability of nanosuspensions (Figure 9) [111]. To prevent
aggregation and preserve the re-dispersibility of the nanosuspensions, the freezing rate is more
important than the subsequent sublimation step [112]. Usually, fast freezing results in smaller ice
crystals and a larger specific surface area of ice which resulting in a decrease in mechanical stress
upon particles. Typically, a higher concentration of cryoprotectant and a faster freezing rate is
better for the redispersibility of the nanosuspensions. During the naproxen nanosuspensions
freeze-drying, when the freezing rate is relatively slow, cryoprotectants such as PEG can migrate
fully into the cryoconcentrated liquid phase, which induced the local concentration of

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cryoprotectant in the liquid phase is relatively high and the aggregation prevention effect can be
enhanced. However, when the concentration of PEG was 40 wt%, no significant aggregation was
found, regardless of freezing rate [113]. Based on these results, Jonghwi Lee group also studied
the mechanism of freeze-drying naproxen nanosuspensions [111]. It was found that irreversible

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aggregation could be occurred during drying rather than freezing, although a proper freezing rate

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is critical. A more homogeneous distribution of the cryoprotectant and the drug nanosuspensions
led to more redispersible powders. Thus, keeping the local concentration distribution of the

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nanosuspensions and cryoprotectant fixed during the freezing step plays a critical role in how the

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freezing rate affects the redispersibility.

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Fig. 9. AFM micrographs showing the effect of the freezing rate on the morphology of dried
powders (PEG 8k): (a) freezing rate = 590 μm/s, (b) 100 μm/s, and (c) 9 μm/s (adopted from
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[111]).
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5.3. Hydrogels
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Beside the above mentioned commonly used solidification methods, hydrogels also is a
practical approach for improving the stability of the nanosuspensions. Buparvaquone, clinically
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against the gastrointestinal persisting parasite Cryptosporidium parvum, is a water insoluble drug.
And it was prepared as nanosuspensions by HPH for oral application. To enhance the stability and
adhesion properties of the nanosuspensions, it was further formulated as hydrogels made from
mucoadhesive polymers, e.g. different types of Carbopol® and chitosan [55]. It was founded that
only a small increasing of the particle size of the bulk population occurred directly after the
incorporation of the nanosuspensions into the hydrogels. The nanosuspensions/ hydrogel systems
were physically long-term stable over a period of 6 months without unchanged particle sizes.
Gupta et al formulated forskolin nanosuspensions using a wet crushing technique stabilized
by Pluronic F127 [114]. By formulating Noveon AA-1 polycarbophil/ Pluronic F127 platforms, at
specific concentrations, it was possible to obtain a pH and thermoreversible gel with a pHgel/Tgel
close to eye pH/ temperature. The addition of forskolin nanosuspensions did not alter the gelation
properties of Noveon AA-1 polycarbophil/ Pluronic F127 and nanosuspensions properties of the
forskolin. Furthermore, the formulation was stable over a period of 6 months at room temperature.

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6. Challenges and future perspectives

The main basic and essential attributes of drug is safety, efficiency and stability.
Furthermore, the safety and efficiency depended on the stability in some sense. And a good

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stability is a warranty for a reliable safety and efficiency. Thus, the stability issue of

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nanosuspensions in drug delivery is a very important and critical aspect of this technology.
Despite the nanosuspensions technology has been studied extensively in recent years, its

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application on pharmaceutics industrial is still restrained. The stability issue of nanosuspensions

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has been highlighted as a major problem for this. The contradiction between the instable inherent
of the nanosuspensions and the needs for meeting the pharmaceutical demanded stability standard
will be promoted the development of nanosuspensions technology itself.

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To our knowledge, this is the first special review focused on the stability issues of
nanosuspensions in drug delivery. However, it was still exiting lots of unknown factors for
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influencing the stability of nanosuspensions. Generally, there are some similarity between
nanosuspensions and micro-suspensions. Therefore, it could be utilizing the explored knowledge
on enhancing the stability of micro-suspensions to improving the stability of nanosuspensions,
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such as decreasing density difference and increasing viscosity. Unfortunately, there are still many
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challenges remaining in the nanosuspensions technology developments, as the significant

physicochemical differences between the nano- and the micro- suspensions.
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In the future, retrieving and establishing the stabilizers usage guideline is a tendency and
requirement for the nanosuspensions technology developments. The stabilization ability of the
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stabilizers, determined the minimal achievable particle size and subsequent physical stability, is a
critical and important factor in nanosuspensions formulation. The consequently physical stability
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of the nanosuspensions is also essential as the irreversible aggregation will impaired the potential
advantages of the nanosuspensions, such as increased dissolution velocity and saturation solubility.
Therefore, the stabilizers play a crucial role in the formation, stability, and overall performance of
nanosuspensions. There are two approaches for the stabilizers developments. The first strategy is
to inquire more usage rules for the present available stabilizers. The most of current reports about
nanosuspensions stabilizers were scattered and no systematically theme issues articles. The second
strategy is to develop more suitable and adjustable new stabilizers such as the biocompatibility
polymers of amino acid, for the nanosuspensions preparing.
Beside the advancements in stabilizers are necessarily, the other aspects also very important,
including but not limited to solidification technique and new preparing approaches, such as
multi-inlet vortex mixing technique (MIVM). Currently, the most related study of
nanosuspensions mainly focused on the efficiency of preparing methods, in vitro dissolution
velocity and solubility, in vivo pharmacokinetics and pharmacodynamics. Therefore, it is

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desiderated to revealing the influencing of preparing approach on the nanosuspensions stability.

And this is also a tendency and requirement for nanosuspensions technology development.
Acknowledgements
This work was supported by the Outstanding Young Scientist Research Award Fund of

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Shandong Province (No: BS2012YY023).

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Figure captions

Fig. 1. SEM of crystallized fenofibrate nanosuspensions prepared at different freezing rates. All
dispersions contain 30% w/w fenofibrate in mannitol. (Magnification left column 1500×;

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magnification right column 10,000×) (adopted from [49]).

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Fig. 2. Trend analysis of particle size stability at the end of 6 week storage at 4 °C with respect to

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enthalpy and LogP of the six drug candidates. And the proposed generic formulation of

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nanosuspensions based on drug properties (adopted from [71]).

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Fig. 3. AFM height image of the ibuprofen surface adsorbed by different stabilizers. A is the bare
ibuprofen surface. B is the HPC adsorbed on ibuprofen surface. C is the PVP adsorbed on
ibuprofen surface. D is the HPMC adsorbed on ibuprofen surface. E is the Poloxamer 188
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adsorbed on ibuprofen surface. F is the Poloxamer 407 adsorbed on ibuprofen surface (adopted
from [19]).
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Fig. 4. TEM pictures of paclitaxel nanocrystals revealed the effects of stabilizer concentration and
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temperature for the nanocrystals thermal stability. (A) paclitaxel/F127 (1:10, w/w) and (B)
paclitaxel/F127 (1:20, w/w), the white spherical structures in the background are micelles formed
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at high concentration of F127. (C) Nanocrystals before 37 °C incubation. Measured size: 176 nm
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(D) longer crystals formed by incubation at 37 °C for 2 h. Measured size: 258 nm (adopted from
[77]).
AC

Fig. 5. SEM images of the celecoxib nanocrystals with different stabilizers following spray-drying
confirmed the influence of stabilizers on the morphological structure of the nanocrystals. (a)
celecoxib API (size bar 10 μm); (b) Tween® 80 nanosuspension (size bar 1 μm); (c and d)
spray-dried nanosuspension stabilized with PVP/SDS (size bars 300, 200 nm respectively)
(adopted from [12]).

Fig. 6. PXRD and SEM micrographs revealed the influences of HPH pressure on the SN 30191
crystallization. A, Form Ⅰ , at low homogenization pressure; B, Form Ⅱ , the initial
crystallization; C, Hydrate (adopted from [89]).

Fig. 7. SEM images of original quercetin API powder (A; bar = 10 mm) and quercetin
nanosuspensions (bar = 1 mm) after 180 days of storage at room temperature (25 °C). B: 5% w/w

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quercetin nanosuspensions produced by HPH; C: 5% w/w quercetin nanosuspensions produced by

bead milling; D: cavi-precipitated nanosuspensions with ethanol (adopted from [80]).

Fig. 8. SEM micrographs of particles prepared by electro-spray drying at various voltages

T
(naproxen nanosuspension concentration = 8 wt%). (a) 0 kV, (b) 1.0 kV, (c) 2.0 kV, and (d) 2.7 kV.

IP
The higher voltage, the less aggregation of the solidified nanosuspensions was observed (adopted
from [103]).

R
SC
Fig. 9. AFM micrographs showing the effect of the freezing rate on the morphology of dried
powders (PEG 8k): (a) freezing rate = 590 μm/s, (b) 100 μm/s, and (c) 9 μm/s (adopted from

NU
[111]). MA
D
P TE
CE
AC

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Graphical Abstract

T
R IP
SC
NU
MA
D
P TE

The graphical AFM height image of the ibuprofen surface adsorbed by different stabilizers directly
CE

revealed why the different stabilizers resulted in different stabilization for the same drug

nanosuspensions.
AC

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