1. REVIEW
A Review: Pharmaceutical and Pharmacokinetic Aspect
of Nanocrystalline Suspensions
DHAVAL A. SHAH,1
SHARAD B. MURDANDE,2
RUTESH H. DAVE1
1
Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, New York 11201
2
Drug Product Design, Pfizer Worldwide R&D, Groton, Connecticut 06340
Received 7 August 2015; revised 23 September 2015; accepted 25 September 2015
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24694
ABSTRACT: Nanocrystals have emerged as a potential formulation strategy to eliminate the bioavailability-related problems by enhancing
the initial dissolution rate and moderately super-saturating the thermodynamic solubility. This review contains an in-depth knowledge of,
the processing method for formulation, an accurate quantitative assessment of the solubility and dissolution rates and their correlation to
observe pharmacokinetic data. Poor aqueous solubility is considered the major hurdle in the development of pharmaceutical compounds.
Because of a lack of understanding with regard to the change in the thermodynamic and kinetic properties (i.e., solubility and dissolution
rate) upon nanosizing, we critically reviewed the literatures for solubility determination to understand the significance and accuracy of the
implemented analytical method. In the latter part, we reviewed reports that have quantitatively studied the effect of the particle size and
the surface area change on the initial dissolution rate enhancement using alternative approaches besides the sink condition dissolution.
The lack of an apparent relationship between the dissolution rate enhancement and the observed bioavailability are discussed by reviewing
the reported in vivo data on animal models along with the particle size and food effect. The review will provide comprehensive information
to the pharmaceutical scientist in the area of nanoparticulate drug delivery. C 2015 Wiley Periodicals, Inc. and the American Pharmacists
Association J Pharm Sci
Keywords: nanocrystals; nanosuspensions; nanoparticles; solubility; dissolution; pharmacokinetics; food interactions; bioavailability;
particle size reduction
INTRODUCTION
Recent advances in synthetic, analytical, and purification
chemistry, along with the development of specialized tools
such as high-throughput screening, combinatorial chemistry,
and proteomics, have led to a sharp influx of discovery com-
pounds entering into development. Many of these compounds
are highly lipophilic, as the in vitro screening techniques place
considerable emphasis on the interaction of compounds with de-
fined molecular targets. In recent years, it has been estimated
that up to 70% of the new drugs discovered by the pharmaceu-
tical industry are poorly soluble or lipophilic compounds. Poor
aqueous solubility is one of the major hurdles in the develop-
ment of new compounds into oral dosage forms, as absorption
is limited by dissolution for these compounds.1
The well-known Biopharmaceutics Classification System
(BCS) is frequently used to categorize pharmaceutical com-
pounds. According to the BCS system, poorly soluble com-
pounds belong to Class II (low solubility, high permeability)
or Class IV (low solubility, low permeability). In another words,
we can also say that Class II and IV compounds provide more
opportunities for the development of newer technologies to
overcome the solubility- or dissolution-related issues based on
chemical and physical properties of the compounds. This per-
ception is widely used and well established within the pharma-
ceutical industry. However, using the BCS system for guidance
in formulation selection may sometimes oversimplify the com-
Correspondence to: Rutesh H. Dave (Telephone: +718-488-1660; Fax: +718-
780-4586; E-mail: Rutesh.Dave@liu.edu)
Journal of Pharmaceutical Sciences
C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
plex nature of drug dissolution, solubility, and permeability.
Poorly water-soluble compounds can possess such a low aque-
ous solubility that the dissolution rate, even from micronized
particle, is very slow. In this case, it is not possible to reach suf-
ficiently high drug concentrations in the gastrointestinal tract
for an effective flux across the epithelial membrane. Other fac-
tors, such as efflux transport or pre-systemic metabolism, can
also negatively influence oral bioavailability.
Therefore, it is recommended to classify compounds into
slightly different categories, as they can show dissolution
rate-limited, solubility-limited, or permeability-limited oral
bioavailability. Butler and Dressman2
designed the “Developa-
bility Classification System (DCS),” as another way to catego-
rize compounds in a more bio-relevant manner. This system dis-
tinguishes between dissolution rate-limited compounds (DCS
Class IIa) and solubility-limited compounds (DCS Class IIb).
In order to select the right formulation approach and to ad-
dress the compound-specific issues with a suitable formulation
type, it is imperative to first understand the bioavailability lim-
iting factors. Selection of the right formulation approach is one
of the key activities for formulators in the pharmaceutical in-
dustry. Key factors include the physicochemical properties of
active pharmaceutical ingredient (API), such as aqueous solu-
bility, the melting point temperature, and chemical stability. In
addition, the formulator needs information about the potency
of the compound and the desired route of administration to de-
termine the type of final dosage form as well as the required
drug load. All these factors can be considered in decision trees,
which are often used in the industry to guide the formulator.
However, there are some biopharmaceutical-relevant as-
pects that need more attention in order to avoid false nega-
tive results. In addition, it is also important to note that there
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES 1

2. 2 REVIEW
is no uniform approach that solves all the formulation-related
problems. Each technology has its own advantages and disad-
vantages. Depending on the formulator’s understanding of the
interplay between the physicochemical properties of the drug,
the special aspects of the various formulation options and the
required in vivo performance, the higher the chance that the op-
timal formulation approach will be chosen. This minimizes the
risk of late failures in the human clinical trials, for example, due
to insufficient or highly variable drug exposures. Compounds
showing dissolution rate limited bioavailability may be referred
to as DCS Class IIa compounds, but they represent only one
part of the BCS Class II compounds. The extent of the oral
bioavailability of such compounds directly correlates with their
dissolution rate in vitro. The fraction of the dose that dissolves
in the lumen is readily absorbed through the intestinal mem-
brane. Consequently, the bioavailability of such compounds can
be improved by any technique that increases the primarily the
dissolution rate. Various formulation approaches are known to
lead to increased dissolution rate and bioavailability, includ-
ing salt formation, the use of cocrystals, particle size reduction,
complexing with cyclodextrins,3
microemulsions,4
and solid dis-
persion technologies.5,6
The formulator has to select the optimal
formulation approach based on the properties of a specific drug
molecule. However, all these technologies have certain limita-
tions and cannot be used as universal formulation techniques
for all the poorly soluble compounds, especially those which
are insoluble in both aqueous as well as non-aqueous solvents.7
To prevent the removal of poorly soluble compounds from the
pharmaceutical pipeline, a broad-based technology is required
for drug molecules that are insoluble or poorly soluble in both
aqueous and non-aqueous solvents. This will have the tremen-
dous impact in discovery sciences and will improve the perfor-
mance of existing molecules suffering from formulation-related
issues.8
In the last two decades, after the introduction of Nano
crystal R
technology, particle-size reduction approaches have
grown to a commercial level. Several formulation ap-
proaches have been reported to formulate the nanoparticles,
such as nanocrystalline suspensions, Poly Lactic-co-Glycolic
acid(PLGA)based nanoparticles, nanosphears, and solid-lipid
nanoparticles. By the virtue of their large surface area (SA)
dc
dt = AD(Cs−C)
h
ln S
S0
= 2MY
DrRT hH = k
√
L/
√
V
Noyes–Whitney Equation Ostwald–Freundlich Prandtl Equation
dc/dt = Dissolution velocity S = Solubility at Temp T hH = Hydrodynamic boundary layer thickness
A = Surface area S0 = Solubility of infinite big particle k = Constant
D = Diffusion coefficient M = Molecular weight L = length of surface in flow direction
Cs = Saturation solubility D = Density V = relative velocity of flowing liquid
C = Drug concentration in U = Interfacial tension
Solution at time t R = Gas constant
h = Thickness of diffusion layer r = Radius
T = Temperature
to volume ratio, nanocrystals provide an alternative method
to formulate poorly soluble compounds. Nanosizing refers to
the reduction of the APIs’ particle size down to the sub-micron
range. Nanosuspensions are sub-micron colloidal dispersions of
discrete particles that have been stabilized using a surfactant
and a polymer or a mixture of both.9
Stabilized sub-micron
particles in nanosuspensions can be further processed into
standard dosage forms, such as tablets or capsules, which are
best suited for oral administration.
It has been studied and observed that the reduction in par-
ticle size in the micron or nano range have a positive impact on
the in vitro dissolution rate, which can be used to predict in vivo
enhancement in bioavailability for poorly soluble compounds.10
Compound-specific properties, such as high melting point, high
log P value and poor aqueous solubility, are required to consider
before the selection of this approach. Therefore, BCS Class II
and IV compounds would theoretically be good candidates for
the nanosizing approach, along with some exceptions, such as
fenofibrate (FBT) (low melting point).11
Drug nanocrystals ex-
hibit many advantages, including high efficiency of drug load-
ing, easy scale-up for manufacture, relatively low cost for prepa-
ration, and applicability to various administration routes, such
as oral, parenteral, ocular, and pulmonary delivery (Table 1).
All these advantages have led to successful promotion of drug
nanocrystals from experimental research to patients’ usage.
The availability of several products on the market shows the
therapeutic and commercial effectiveness of the approach.12
The pioneering work of many academics and industrial re-
searchers has laid the foundation for broad utilization and ac-
ceptance of this approach within the field of pharmaceutical
sciences.
By definition, nanosizing is particle-size reduction to 1 and
1000 nm. Because of their small size, these particles can vary
distinctly in their properties from micronized drug particles.
Similarly to other colloidal systems, drug nanocrystals tend to
reduce their energy state by forming larger agglomerates or
crystal growth, which is why they are often stabilized with sur-
factants, stabilizers, or with a mixture of both. Reduction of the
particle size to the nanometer range results in a substantial
increase in SA (A), thus, this factor alone will result in a faster
dissolution rate as described by Noyes–Whitney.13
In addition,
the Prandtl equation shows that the drug nanocrystals showed
decreased diffusional distance “h”. This further enhances the
dissolution rate. Finally, the concentration gradient (Cs − Cx) is
also of high importance. There are reports that drug nanocrys-
tals have shown increased saturation/thermodynamic solubil-
ity (Cs). This can be explained by the Ostwald–Freundlich
equation14
and by the Kelvin equation.15
It is still not clear to what extend the saturation solubility
can be increased solely as a function of particle size. Most prob-
ably the increased solubility of drug nanocrystals is a combined
effect of nanosized drug particles and solid-state effects caused
by the particle fractionation during the process. A number of
authors have reported improvement from a 10% increase in
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

3. REVIEW 3
Table 1. Advantages of Nanocrystals in Different Route of
Administration
Route Advantages
Oral r Increase bioavailability
r Decrease in fed/fast variations
r Increase rate of absorption; decrease in Tmax
and increase in Cmax
r Quick and easy to formulate
Parenteral r About 100% bioavailability can be achieved if
given as an IV formulation
r Targeting drug delivery
r Avoidance of organic solvent, surfactants, pH
extremes
Pulmonary r Used in nebulizer as a liquid solution or dry
powder
r A single drop can contain many nanoparticles
r Increase the concentration and or loading of
nanocrystalline dispersion
saturation solubility to several folds using different
approaches.16–20
Below are the established equations to de-
scribe nanocrystals and their physicochemical properties.
Advantages of nanocrystals over conventional and special
drug delivery systems:
1. Because of high surface enlargement factor in nanocrys-
tals, there is an increase in the dissolution rate as well
as a modest increase in saturation solubility as compared
with micronized particles.
2. With a size range in nanometers, it can be injected as a
IV to get 100% bioavailability.
3. Dose reduction and patient compliance.
4. Lessen or eliminate the food effect on bioavailability.
5. Targeted drug delivery either by transcellular or intra-
cellular uptake.
6. Molecule can be delivered via a required route with ease
in scale up.
This review focuses on the various established approaches
for the formulation of nanocrystals, the different published an-
alytical methods applied for thermodynamic solubility deter-
mination, assessment of dissolution properties and dissolution
rate enhancement upon nanosizing, the effect on pharmacoki-
netic (PK) properties such as bioavailability, the area under
curve (AUC), and the half-life due to size reduction as well as
future research opportunities.
FORMULATION APPROACHES FOR NANOCRYSTALS
Before the first top-down processes were developed (i.e., tech-
niques reducing the size of larger crystals by means of attrition
forces), nanosized drug particles were produced using a sim-
ple precipitation approach known as solvent–anti-solvent ad-
dition technique. It is also referred as one of the “bottom-up”
approaches. However, it is often difficult to control the particle
growth/crystal growth using this technique as well as to scale
up by maintaining all the parameters constant. Therefore, it
was suggested to perform the precipitation step in conjunction
with immediate lyophilization, or spray-drying, in order to re-
duce the risk of crystal growth.
Top-Down Approach
There are two basic approaches which are well established for
the formulation of nanocrystals:
1. Top down: Involves the mechanical reduction of the par-
ticle size by wet media milling or high-pressure homoge-
nization (HPH).
2. Bottom up: Involves the generation of nanosized particles
from dissolved molecules by means of precipitation.9
Top-down methods can be further divided in to two
approaches—homogenization and attrition wet media milling.
Attrition Wet Media Milling
This technology was developed at the Pharmaceutical Research
Division of Eastman Kodak (Sterling Winthrop, Inc.), which
was set-up as NanoSystems LLC and later acquired by Elan.
An active drug substance is dispersed with an aqueous solution
in which the stabilizers were pre-dissolved. As the surface of
nanocrystals is highly cohesive and has high surface energy,
it should be stabilized by a single or mixture of stabilizers.
Stabilizers can be ionic or stearic and can be used as a single
and/or in a combination of polymeric as well as surfactant sta-
bilizers. This solution is poured in the grinding chamber along
with spherical beads/balls while the beads are rotated at very
high speed. It is believed that because of the attrition between
molecules’ surface and surface of the beads, particle size reduc-
tion occurs; the beads/balls serving as a milling media. Beads
are available in various sizes and are of different materials,
but generally are made of glass, zirconium oxide, or polymeric
material. The type of material the beads are made of is a crit-
ical factor as they can interact with the active drug substance.
There is a fair chance that an impurity related to the material
of beads may contaminate the final product. Yttrium-stabilized
zirconium oxide is the most widely used type of bead by ma-
jor pharmaceutical companies because in most cases, it does
not interact with active drug substances. Although expensive,
these beads are the best alternative to avoiding impurities in
the final formulation.21
The size of the beads has a direct relationship with the de-
sired particle size range in the formulation of nanocrystals.22
The usual duration for conventional milling using overhead
stirring is somewhere between 3 and 12 h. Certainly, these pa-
rameters can change from molecule to molecule. Milling should
be stopped once the desired particle size range is achieved. The
rotational speed of the milling media is also a critical parame-
ter. With the too slow speed, the beads cannot rotate efficiently
and milling cannot be performed accurately, and with the too
fast speed, the evenly rotating balls may remain at the upper
surface of the media and milling does not take place. With a
systematic study by trial and error the formulator selects the
stabilizers, as well as other milling parameters and optimizes
them in order to achieve the desired particle size range and
stability. The final product characteristics can vary, depend-
ing on the amount of beads, the ratio of active drug substance
to the amount of beads, the ratio of concentrations of active
substance to the stabilizer, milling time, milling temperature
as well as milling duration.23
This method is simple, inexpen-
sive, and easily scalable. The only drawback associated with
this technology is the contamination related to the beading
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

4. 4 REVIEW
material. That aside, several products have successfully
reached the commercial level using this technology.
High-Pressure Homogenization
There are several established methods for the formulation of
nanocrystals using the homogenization approach. The microflu-
idization technology (Insoluble Drug Delivery-Particles IDD-
PTM
Technology), Dissocubes R
technology, and Nanopure R
technology are examples of the methods that fall under this
category. Microfluidizers are known as high shear fluid pro-
cessors that are unique in their ability to achieve monomodal
particle size reduction. It reduces particle size by a frontal colli-
sion of fluid streams under pressure of up to 1700 bar.24
At very
high pressure, collision and cavitation occur. The major draw-
back associated with this method is that it requires at least
50–75 cycles to achieve the desired nanometer size range. This
makes the method more tedious and relatively more time con-
suming as compared to milling. Dissocubes R
technology works
with piston gap homogenizer, which was developed by Muller
and his colleagues. In this method, a crude aqueous suspension
of active drug substance and stabilizer is forced through a tiny
hole, which can reach a pressure of up to 4000 bar. The width
of the homogenization gap is adjustable, which is typically in
the micrometer range.
Compared with wet media ball milling, there are fewer
chances to generate impurities with HPH. The negative aspects
of using this method are cavitation, which causes mechanical
wear, as well as noise, although fragmentation is a beneficial
effect associated with cavitation. The main source of impurity
comes from the wearing out of equipment parts. Almost all ma-
chine parts are made of stainless steel, which leads to a very low
impurity level when the nanosuspension is prepared using the
HPH. Krause and Muller25
carried out a comparative study and
observed a negligible amount of iron impurities in the nanosus-
pension formulated with 20 cycles at 1500 bar. Wear and tear
occurs only when very hard material is processed through the
piston gap. Using stainless steel material can also lead to wear
and tear as the new type of homogenization valves used today
are made of ceramic tips which are able to withstand the harsh
processing conditions.26
Homogenizers vary in size from a small
scale to large scale production.27
Many research studies have
reported minimal growth of microorganisms as a result of the
HPH process.28
These improve the shelf life of the nanosuspen-
sion and avoid the need for further studies that are required
if it is administered orally. However, it is not a rule of thumb,
the HPH is generally used for relatively soft material and bead
media mill is used for relatively harder or harsh material.
Combinative Approach
In order to proceed with both the top down technology (wet me-
dia milling, HPH) micronized powder is required as the starting
material, which leads to a long process time. In order to over-
come this drawback, a combinative formulation approach was
developed. The combinative approach was first developed and
introduced by Baxter Inc. as NanoedgeTM
technology. Today five
combinative methods have been successfully developed.
1. NanoedgeTM
—microprecipitation + HPH
2. H69—microprecipitation immediately followed by HPH
(minimization of time between two steps in order to pro-
duce even smaller crystals)
3. H42—drug pre-treatment by means of spray-drying fol-
lowed by standard HPH
4. H96—Freeze drying combination with HPH
5. CT—Media milling followed by HPH
In the microprecipitation stage, the drug is usually dissolved
in a suitable organic solvent that is miscible with water. The
drug solution is then added to an aqueous solution in which
stabilizers have been pre-dissolved. The drug solution is added
in a controlled manner to prevent inadequate crystal growth.
After the microprecipitation step, precipitates are converted
into more stable crystals in the nanometer size range with the
help of top down technologies (i.e., HPH, media milling, and
sonication). The amount of residual content in the final prod-
uct is the major concern while using a combinational approach
during scale up. The presence of organic solvent can alter the
physicochemical properties of the active drug substance.29
It
may also be responsible for the Ostwald’s ripening. To prevent
this from happening, an alternative method was developed by
Salazar et al.21
known as H 42 and H 96 technology. H42 uses
the spray drying of the microprecipitated solution that was de-
veloped with the bottom-up approach, and then followed by
HPH. In the case of H96, it employs the freeze drying of the mi-
croprecipitated solution, followed by a top-down approach. In-
deed, on the one hand, this method has more advantages than
any single step conventional method, but on the other hand,
any additional steps in the procedure require more careful and
more extensive research, and control of additional parameters,
which will increase the cost of the end product development. To
date, no product has been developed and marketed using this
technology, but research papers have been published for the
formulation or production of nanocrystals using the combina-
tive approach. Among these, the top-down approaches are more
convenient because of the ease of being able to govern the parti-
cle size range as well as the ease of scaling up. Because of these
benefits, several products have been successfully launched to
the commercial level.
Bottom-Up Approach
This method is also known as the precipitation approach.
Hydrosols30
and Nanomorph31
techniques are examples of the
bottom-up approach. The particles generated by Nanomorph
technology are amorphous in nature, which give an advantage
of both a higher supersaturation and a higher dissolution rate.
It is well known that amorphous systems are high energy sys-
tems; therefore, because of their high rate of crystallization, un-
controllable crystal growth occurs, which leads to a reduction in
solubility and eventually, reduction in bioavailability. Although
both technologies are scalable, they require the control of dif-
ferent parameters, such as temperature and the stoichiometry
of the solute, solvent, and the stabilizer.
UNDERSTANDING SOLUBILITY BEHAVIOR AND
METHODS OF DETERMINATION
Several research papers have discussed the impact of solubility
and particle size on the PK performance of nanocrystals. Some
of the literature has reported that the generation of surface cur-
vature and crystal defects on the particle surface have an enor-
mous impact on its solubility behavior. Another possible cause
might be the development of high energy surfaces through
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

5. REVIEW 5
attrition during particle size reduction. According to the litera-
ture, solubility may range from one-fold to several fold, based on
particle size.17–19
Bioavailability enhancement associated with
nanocrystalline API is attributed to an increase in the dissolu-
tion rate because of the enlargement of SA and some increase
in solubility based on particle size. This solubility enhance-
ment should be in fair agreement with what would be expected
based on the Ostwald–Freundlich equation. A change in solu-
bility is more significant when particle size is reduced to below
100 nm, which can also be described by the Ostwald–Freundlich
equation. Rapid dissolution associated with a nanoparticulate
system is clear evidence of a generation of transient super sat-
uration of a solution compared with the bulk solubility of a
stable crystal form. In the case of crystalline nanoparticles, the
degree of supersaturation is low compared with high energy
amorphous solids, as particle size has limited impact on satu-
ration solubility.
Determining accurate solubility is vital to characterize the
effectiveness of the formulation. There are several challenges
associated with the accurate determination of solubility of any
formulation, as it varies case by case. Accurate measurement
is significantly more complicated in the case of nanoparticulate
systems, as they have the tendency to remain in suspended
form in the solution after using conventional approaches. It is
almost impossible to visualize the presence of nanoparticles in
the filtrate with the naked eye. The intrinsic solubility of poorly
soluble compounds is extremely low in number; therefore, the
presence of a couple of undissolved particles can lead to a signif-
icant error in measurement. While reviewing the literature for
determining solubility by a separation-based method, we have
found the absence of a validated universal method for accurate
solubility determination. This makes it even more challenging
when dealing with particle size in the nanometer range, as com-
pared with the micronized or bulk particles. The following are
the general challenges associated with solubility determination
of the nanoparticulate system.
1. No standard method is available in the literature for sol-
ubility determination,
2. Difficulty in separation of the dissolved and undissolved
nanocrystals/particles because of smaller size.
3. Confirmation that equilibrium is attained or not.
4. Reproducibility of results.
5. Validation of method for accuracy.
In addition to the above challenges, one also has to consider
other process parameters which vary with the physicochemical
properties of the active drug substance for solubility determina-
tion. For instance, if the API is weakly acidic or basic, then the
pH of the solution plays an important role. It is difficult to deter-
mine whether or not equilibrium is attained in this particular
case. Several researches have published different approaches
for the solubility determination of nanocrystals. Although nu-
merous methods for the separation of dissolved and undissolved
nanoparticles have been reported in the literature, these are the
most common approaches used described in Figure 1. An aque-
ous solubility determination by separation-based approach is
widely accepted, has been used in industry and academia for
many decades, and is the most convenient way to determine
solubility. Typically, it is a two-step process: initially, an excess
amount of drug is dispersed in an aqueous or buffer solution.
The equilibrium is established by shaking or stirring the solu-
tion at a specific rpm for a specific time and temperature, at
which we want to determine the solubility. Usually the sam-
ples are withdrawn after 24 and 48 h. Samples were either cen-
trifuged or filtered from syringe filters. A sample analysis was
performed using the HPLC and UV. In the case of crystalline
nanoparticles, the research articles listed in the Table 2 have
reported the solubility determination data for nanosuspensions
and nanocrystals by utilizing a separation-based approach as
their primary method for solubility determination. The most
commonly reported approaches for solubility determination of
nanoparticles are by shake-flask method at a specific temper-
ature. Most of them have overestimated the thermodynamic
solubility associated with nanocrystals, which is why it is im-
portant to consider some additional factors during solubility
determination when particle size is reduced to nano from mi-
cron range. The selection of appropriate pore size filters with
respect to the particle size of crystals, and the selection of an
appropriate spectroscopic analytical method, is the key factor
that needs to be taken in to consideration for accurate solubility
determination of crystalline nanoparticles.
Bernard Van Eerdenbrugh reported that the UV spectra is a
reliable tool to determine the concentration of micronized par-
ticles, however, it is not a reliable tool for the determination
of the concentration of nanosuspensions, as it is overestimat-
ing the actual solubility data. With particles in the nanome-
ter range, they itself absorbs the UV light. Therefore, the ab-
sorbance data are the mixture of the dissolved and undissolved
nanosized particles.32
Today, the measurement of the dissolved
drug concentration using an in situ UV probe is the preferred
noninvasive method because of its sophistication in terms of
contingency and its ability to record data from the start of dis-
solution. However, absorption of light from particles is size de-
pendent and it is having a great influence on smaller particles.
With felodipine as a model drug, an observation has been made
that both nanoparticulates of felodipine and free felodipine in
the solution absorb light in a similar way, which results in an
overestimation of dissolved concentration than what was actu-
ally dissolved.33
The results were also dramatic, even for the
second derivative of UV spectra. Moreover, the generation of
nanoparticles occurs when working with nanosuspensions or
supersaturated systems, so caution should be taken.
The solubility associated with crystalline nanoparticles is
moderately higher (10%–15%)16
compared with what is re-
ported in Table 2.34–44
In the case of indomethacin, the reported
solubility enhancement was nearly twofold higher. The same is
true in the case of oridonin,36
reccardin D,38
and simvastatin39
where reported solubility was substantially higher as compared
with bulk crystal solubility. One possible reason behind this
misleading data may be the use of an inappropriate separating
method and/or analytical method. Nanoparticles with an aver-
age size of 200–400 nm can easily pass through 0.22 or 0.45 :m
pore sized filters. Determining the concentration of such a fil-
tered solution using UV leads to a further over-estimation of
actual data because of the mixture of absorbance of both the
undissolved and dissolved particles. Moreover, centrifugation
at moderate speed of about 10,000–30,000 rpm is not suffi-
cient to suspend particles in the nanometer range, therefore,
the resultant supernatant contains a mixture of dissolved and
undissolved particles. It is noticeable that care should be taken
in choosing syringe filters for appropriate pore size. Juene-
mann et al.45
were able to show in their study a differentiation
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

6. 6 REVIEW
Table2.ExperimentalSolubilityDeterminationwithSeparation-BasedApproach
DrugMethodFiltration
ParticleSizeand
AnalyticalInstrumentReportedSolubilityReference
Indomethacin
(IND)
Shake-flaskat25°C0.2:mUVNSa:5.86±1.2mg/100mL34
Physicalmix:2.30±0.51mg/100mL
Poly1:0.91±0.26mg/100mL
Poly2:1.44±0.34mg/100mL
Indomethacin
(IND)
Shake-flaskfor12hinacetate
bufferpH5
NMbUVIND–physicalmix:4.89±0.18:g/mL35
P.size:80:mNano-IND/F68:6.43±0.06:g/mL
580±30nmNano-IND/F127:4.80±0.0:g/mL
580±20nmNano-IND/polysorbate:80:10.9±1.54:g/mL
Notdetermined
Oridonin(ORI)Constant-temperatureshaker
at37°Cand100rpmin
phosphatebuffersolution
(pH7.4)
0.22:mmicropore
film
UVORI(commercial):99±2:g/mL36
P.size:200–400nmORInanocrystal:170±10:g/mL
However,authorreportedslowdecreasein
solubilityafterachievingsupersaturationfor
NC.c
Candesartan
cilexetil
24hstirring,followedby
centrifugationat25,000rpm
inphosphatebuffer
containing0.7%Tween20
(pH6.5)
0.2:mUV
P.size:223.5±5.4
Bulk:125±6.9:g/mL
NC:2805±29.5:g/mL
22.44-foldimprovement
37
RiccardinDStirringinPBS(pH7.4)buffer
at25°Cand100rpm
0.22:m
microporous
membranefilter
HPLC
P.size:184.1±3.15nm
P.size:815.37±9.65nm
Bulk:0.6192±0.0245:g/mL38
NS(evaporativeprecipitationintoaqueous
solution):242.1±12.1:g/mL
NS(microfluidization):31.5±1.9:g/mL
SimvastatinPowderform:shake-flask
methodat37°C,
0.22:mWhatman
filter
Supernatant
UV
P.size:300.3nm
Solubilityenhancement:36.14-fold39
NS:centrifugationat
10,000rpm
FenofibrateShakeflaskmethod,suspension
equilibratedat37°Cfor72h
0.22:mmembrane
filter
HPLCSolubility40
NS:P.size:606nmConc.SDS%(W/V)Bulk-solubility
(:g/mL)
0.00.34±0.03
0.11.42±0.10
0.1511.95±0.16
0.226.38±0.39
0.376.11±0.38
0.4131.95±1.68
0.5183.09±2.17
0.7290.43±5.70
Continued
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

7. REVIEW 7
Table2.Continued
DrugMethodFiltration
ParticleSizeand
AnalyticalInstrumentReportedSolubilityReference
Atorvastatin
calcium
Shakeflaskmethod,for24h
and37°CinDIwater
0.1:mmembrane
filter
UV41
P.size:commercial:38.3±
0.6:m
142.2±0.5:g/mL
TurraxR
=21.5±0.03:m185.1±1.2:g/mL
HPHd:3.12±0.05:m299.8±0.6:g/mL
HPH(20cycles@1500bar:
=0.446±0.02:m
386.5±0.7:g/mL
MeloxicamShakeflaskmethod,for24h
and37°CinDIwater,stirred
samplewerefurther
centrifugedat10,000rpmfor
15min
0.22:mnylon-
membrane
filter
UV42
P.size:Raw:4.4±0.50:g/mL
Raw:46.39±7.37:mPhysicalmixture:5.83±0.62:g/mL
Sonicated:0.259±0.03
:m
Spray-driedNC:21.84±0.78:g/mL
Sonicated+HPH:0.212±
0.04:m
Spray-dried:0.178±0.02:g/mL
LuteinSampleswerekeptonshaker
100rpmand25°Cdistilled
water(pH=5.5)anddistilled
watercontainingsurfactant
solution(0.05%,w/w
PlantacareR
2000,pH=
10.5).Sampleswere
centrifugedat23,800gfor2h
0.2:mfilterUVDIwater:<0.054:g/mL
DIwater+surfactant:0.54:g/mL
nanocrystal:upto14.3(>264-fold
thanwaterand>26.3-foldwater
withsurfactant)
43
P.size:429nm
QuercetinSampleswerekeptonashake
at37°Cand100rpm.Sample
werewithdrawnandtransfer
toultrafreetubewithcutoff
of10kDaandcentrifugedat
20,000gfor30minat4°C
NMHPLCBulk:10.28g/mL44
P.size:PM:16.42g/mL
Dried-EPAS:282.6±50.3
nm
EPAS(evaporativeprecipitationinto
aqueoussolution):422.4g/mL
HPH:213.6±29.3nmHPH:278.6g/mL
a
NS,nanosuspensions.
b
NM,notmentioned.
c
NC,nanocrystal
d
HPH,high-pressurehomogenization.
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

8. 8 REVIEW
Figure 1. Reported approaches for the solubility determination.
between suspended submicron colloidal particles and molecu-
larly dissolved particles. It has been reported that the results
of solubility and dissolution of nanocrystals are comparable
with each other if the analysis is performed with filters having
pore size ࣘ 0.1 :m. Using larger 0.2 and 0.45 :m filters, the
nanocrystalline system shows apparent supersaturated behav-
ior because of the combinatory effect of dissolved and undis-
solved nanocrystalline particles. In other words, it has been
demonstrated that filters with a bigger pore size may not be
sufficient enough to hold back the colloidal particles. An ex-
cellent demonstration has been reported by carrying out the
study of a selection of appropriate filters and their impact on
observed in vitro results. It was also compared with an in silico
model for more precise justification of the experimental data.
Overall, the conclusion and recommendation has been made
for using filters with smaller pore sizes (i.e., 0.1 and 0.02 :m)
when dealing with nanoparticles.
Kinetic solubility determination by nephelometric or turbidi-
metric methods during early screening of the drug molecule was
introduced earlier by Bevan and Lloyd.46
Bernard Van Eerden-
brugh critically evaluated different methods for the solubility
determination of nanocrystals using four model compounds:
itraconazole, loviride, phenytoin, and naproxen. The data ob-
tained show that separation-based methodologies were not suf-
ficient to determine solubility, as the data were not in fair agree-
ment with the Ostwald–Freundlich equation. Noninvasive
analytical techniques, such as light scattering and turbidime-
try, were found to be more reliable for appropriate understand-
ing of the solubility behavior of crystalline nanoparticles. In
the case of amorphous nanosuspensions, Lindfors et al.33
have
determined the solubility by plotting scattering intensity with
the drug concentrations. Presence of excessive stabilizers in
nanosystems also tends to generate the scattering of intensity.
Hence, the method that Lindfors demonstrated is not accurate
enough because of the summation of scattering intensities that
are generated by dissolved nanoparticles and micelles. In or-
der to eliminate this, Van Eerdenbrugh et al.16
have chosen
the point of intersection at which scattering is exclusively due
to the dissolved drug concentration and determine the solu-
bility. Measures solubility data by scattering, for loviride was
comparable to bulk solubility data. It was not feasible to
determine solubility for itraconazole from the turbidimetry
method, as the method cannot be distinguished because of very
low solubility. Solubility determination for phenytoin can be
determined precisely with both light scattering and turbidime-
try. In the case of naproxen, determined solubility values were
slightly less than the unmilled compound but the results were
within the standard margin of error. Hence, this shows that
both turbidimetry and light scattering methods can be applied
to obtain more realistic solubility data for crystalline nanopar-
ticles. Scattering can be used more precisely for compounds
that have low scattering intensities and turbidimetry can be
applied on compounds having higher scattering intensities.
Moreover, the solubility enhancement was in fair agreement
with the theoretical prediction from the Ostwald–Freundlich
equation. For the theoretical calculation, the interfacial ten-
sion was estimated (interfacial tension of typical pharmaceu-
tical API ranges between 5 and 50 mN/m) as described in the
reference literature.47
Experimental data and theoretical pre-
dictions were also in agreement with the Ostwald–Freundlich
equation, suggesting that solubility enhancement should be
marginal in the case of crystalline nanoparticles and both the
light scattering and turbidimetry are good noninvasive meth-
ods for the solubility determination of nanocrystals. However,
there are certain assumptions and limitations associated with
these analytical methods. In most cases, the dynamic light scat-
tering is used to measure the light scattering intensity. The
instrument assumes each particle as a sphere and carries out
the determination. When the particles are not spherical ini-
tially or if the particles tend to change shape during disso-
lution or solubilization, they may have different results than
those measured by the instrument. Therefore, the impact of
particle shape is a factor that also needs to be considered, as it
plays major role during dissolution and solubilization. Anhalt
et al.48
has reported a method for the solubility determination
by real time measuring the light scattering. The main focus was
to determine the equilibrium solubility and dissolution rate of
a crystalline nanosuspension with different particle size using
FBT as a model compound. The solubility results of the light
scattering method were fair enough to justify it as a reliable
analytical tool for the solubility measurement. Reported sol-
ubility enhancement for nanocrystals was around 10%–15%,
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

9. REVIEW 9
Table 3. Alternative Approaches for Accurate Solubility Determination
Analytical Method, Compound and Particle Size Solubility Determination Solubility Enhancement Ratio Reference
Light scattering 48
Fenofibrate 8.69 ± 0.78 :g/ml
140 nm 10.38 ± 0.01 :g/ml 1.19
270 nm 8.70 ± 0.24 :g/ml 1.00
1070 nm 9.62 ± 0.50 :g/ml 1.11
Light scattering and turbidimetry 16
Loviride (162 nm) 0.0108 mg/mL 1.10
Scattering – 0.0119 mg/mL
Itraconazole (220 nm) 0.0047 mg/mL 1.15
Scattering – 0.0054 mg/mL
Phenytoin (406 nm) 0.0667 mg/mL 1.07
Scattering – 0.0711 ± 0.007 mg/mL
Turbidity – 0.07 ± 0.0034 mg/mL 1.05
Naproxen (288 nm) 0.2116 mg/mL 0.97
Scattering – 0.2053 ± 0.003 mg/mL
Turbidity – 0.2083 ± 0.0024 mg/mL 0.97
Separation-based methodology (ultracentrifugation, filtration—0.1 :m, 0.02 :m, and dissolution) 51
Griseofulvin-micro 7.63 ± 0.89 :g/mL 1.10
362 nm 8.40 ± 0.25 :g/mL
122 nm 9.99 ± 0.15 :g/mL 1.30
Compound X-micro 65.17 ± 1.58 :g/mL 0.97
238 nm 63.41 ± 1.26 :g/mL
93 nm 89.06 ± 6.36 :g/mL 1.36
Fenofibrate-micro 0.74 ± 0.27 :g/mL 1.11
290 nm 0.82 ± 0.26 :g/mL
Celecoxib-micro 1.00 ± 0.03 :g/mL 1.11
341 nm 1.11 ± 0.03 :g/mL
which justifies the previously reported solubility data by Van
Eerdenbrugh.16
However, one should consider certain limita-
tions before utilizing this light scattering method. Most impor-
tantly, the sample has to be ultra clean; the presence of any dust
and debris may lead to the generation of scattering intensity
that affects to the observed data. The use of a plastic cuvette is
also a potential source of error. Moreover, as light scattering is
less sensitive to small particles, the results are slanted towards
the larger sized particles as they have a tendency to generate
more scattering of light. Besides these methods, other analyti-
cal techniques like potentiometry and pulse polarography have
also been reported for real time solubility measurement.49,50
However, these methods are not universal and can be used for
a fewer number APIs with certain properties (i.e., electroac-
tive). In the case of potentiometric measurement, each time a
specific electrode has to use for the specific API.
In general, separation-based methods are universally ac-
cepted as they do not require a high level of experimental skill.
Murdande et al.51
has reported three different approaches (ul-
tracentrifugation, ultracentrifugation with filter, dissolution) to
determine the accurate solubility. Ultracentrifugation was used
after optimizing different parameters such as speed, time, and
temperature for the satisfactory separation of dissolved and
undissolved nanocrystals. Syringe filters were also used (i.e.,
0.1 and 0.02 :m) based on the particle size to determine sol-
ubility from the supernatant of the ultra-centrifuged sample.
In addition, dissolution data at the end of the experiment was
also evaluated for the total dissolved concentration based on the
theoretical knowledge that nanocrystals should reach the satu-
ration level at equilibrium. Results from all three methods were
similar and in fair agreement with the Ostwald–Freundlich
equation. The results from the solubility determination using
the separation-based approach described above are in Table 3.
Interfacial solubility (the concentration in a boundary layer of a
spherical particle) plays an important role. As the particle size
decreases, the SA increases proportionally, along with the in-
terfacial tension. Sun et al.52
have determined the equilibrium
solubility of coenzyme Q10 nanocrystals and bulk drug in three
different dissolution media, which were mixtures of different
concentrations of tween 20 and isopropanol. Solubility usually
leads via diffusion and the driving force would be the difference
between the solubility at the boundary layer and the bulk drug
concentration. Based on observations, they had also proposed
a solubility model for nanocrystals and bulk drugs.52
Another separation-based approach is the determination of
solubility and/or drug release with the application of equi-
librium dialysis. The published research has applied this
concept, mainly for the nanoparticle-based formulation of
large molecules and a few for small molecules, and de-
termined the equilibrium solubility and drug release from
nanoparticulate formulation. The most common approaches in-
clude: sac dialysis,53,54
side by side dialysis,55–57
and reverse
dialysis.58,59
In dialysis, separation is achieved by means of
a semi-permeable membrane using molecular weight cut-off
membrane. Assuming that the dialysis membrane is perme-
able to free API only, Frank et al.60
reported the quantification
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

10. 10 REVIEW
of molecularly dissolved, poorly soluble drug by using the equi-
librium dialysis method and determined the apparent solubility
(molecularly dissolved API + miscellany solubilized drug). The
solubility of the drug in both the Hanks Balance salt solutions
and the supplementary salts (HBSS++) and fasted-state sim-
ulated intestinal fluid (FaSSIF) buffer were nearly the same
within the margin of error.60
It is well known that nanoparti-
cles have a rapid release dissolution kinetics. Efforts have been
made to determine the interplay of the diffusion rate, size of
the molecular weight cutoff (MWCO) membrane, and concen-
tration difference of the donor and receiver compartment by
Moreno-Bautista and Tam.61
Decreasing the dialysis cassettes
to smaller MWCO showed a reduction in the diffusion rate. The
compounds having a molecular weight of around 200–400 Da
showed a diffusion rate profile several times higher with 10 kDa
membrane compared to 2 kDa membrane MWCO. The cassettes
having a pore size smaller than the drug molecule should be
suitable for the evaluation of the dissolution rate of nanopar-
ticles. Furthermore, the release profile from the dialysis cas-
settes failed to catch some necessary events, such as the burst
effect and lag time, which leads to a question the suitability of
the method for true discrimination of the rapid release kinetics
of submicron size colloidal particles. This situation prompted
Zambito et al.62
to ask question whether the dialysis is a re-
liable method for studying drug release from nanoparticulate
systems. Their study concluded that for nanoparticles, the dial-
ysis method was insufficient in expressing the discrimination
and founded results were deceptive. Because of a reversible in-
teraction between the drug and dispersed nanoparticles, the
rate of permeation through the dialysis membrane was neg-
ligible as compared with the plain drug solution. Hence, the
kinetic release was found to be far less and unrealistic. With
such a study, the release kinetic is governed by the dialysis
membrane (not dependent on the drug release from colloidal
particles), which makes it unreliable for the in vitro predictions
and means the data may not be very discriminative.
DISSOLUTION
Dissolution is known as a significant tool for the analysis of
pharmaceutical formulations. Indeed, it has somewhat poten-
tial to predict the in vivo performance. Prediction of the extent
of absorption can be made from the rate of drug release in the
gastrointestinal tract.63
A drug candidate has to pass through
some pre-requisites steps before being absorbed into the sys-
temic circulation. Dissolution is one of the essential steps for
effective absorption of a drug candidate and is a key parame-
ter for assessing the onset of action of an oral dosage form. As
per the well-known BCS, classification compounds belonging to
Class II and IV are poorly soluble in nature, especially ones
belonging to Class II, which have a dissolution rate of limited
absorption because of their low aqueous solubility.64
In another
words, the rate of absorption is proportional to the rate of disso-
lution in the gastrointestinal tract. For a given drug moiety that
dissolves in the gastrointestinal tract, its dissolution can be tai-
lored by either generating amorphous systems or reducing the
particle size to the micron or submicron level (i.e., nanometer).
The motivation for the development of nanosystems has been
generated from the increased number of new chemical entities
with poor aqueous solubility. For such low aqueous-soluble com-
pounds, even micronization is not sufficient enough to eliminate
the problem. The reduction of particle size leads to an increase
in the SA of the drug particle. The SA enhancement factor from
micron to nanoparticle is about 10-fold. Thus, significant en-
hancement in SA has a positive impact on the dissolution rate
of a drug particle. This positive effect on the dissolution rate
can create a higher concentration difference between the gut lu-
men and the blood, which increases the absorption via passive
diffusion and leads to an improved therapeutic response. Direct
proportionality of the rate of dissolution with respect to specific
SA has been well documented by the Noyes–Whitney equation.
As a follow up to this, Nernst–Brunner and Danckwerts also
proposed modified equations to describe the dissolution behav-
ior a solid powder.
Noyes − Whitney Equation :
dM
dt
= k(Cs − C) (1)
Nernst − Brunner Equation :
dM
dt
= S
D
h
(Cs − C) (2)
Danckwerts surface − renewal model :
dM
dt
= S
√
DP (Cs − C)
(3)
This section will provide an overview of the different estab-
lished methods to assess the dissolution for nanocrystals. As
of now, it is well understood that particle size is inversely pro-
portional to SA and the dissolution rate has a direct relation-
ship with the change in SA. The major challenge is to develop
a discriminative dissolution method for poorly water-soluble
drugs. Earlier reports have indicated that there is an alteration
of wetting behavior with particle size reduction. In the case
of nanoparticles, the wetting phenomenon is more prominent,
which leads the dissolution rate to increase more quickly. More-
over, the reduction in the diffusional distance upon size reduc-
tion generates a moderate spring effect that quickly achieves
supersaturation level with respect to particle size during dis-
solution (Fig. 2). Therefore, the traditional methods are inad-
equate to determine the actual dissolution rate enhancement
for nanoparticles which intensify the need for an appropriate
method to perform the dissolution study of nanoparticles.
Sink Condition (Conventional) Dissolution
Sink condition dissolution condition is defined as the volume
of the dissolution medium which can dissolve more than three
times the amount of the dose used in the dissolution study.65
In most cases, the dissolution study is performed using a USP
type – I or II apparatus. Usually for a study of a drug release
profile, the dissolution is carried out in 900 mL of dissolution
media (i.e., in a different pH or in deionized water) at 37 ± 0.5°C
temperature and by rotating at a specific rpm (i.e., 100 rpm).
Samples are withdrawn at specific time intervals throughout
the dissolution study. After each sample withdrawal, the same
amount of the fresh dissolution media, which is equilibrated at
the same temperature, is introduced to maintain perfect sink
condition. In most cases, samples will be filtered prior to anal-
ysis. However, for the nanoparticles, sink condition dissolution
will provide good qualitative and less quantitative information
regarding the dissolution behavior of the formulation. So, the
comparison of dissolution profiles between nanosuspension and
microsuspension will show the impact of particle size on disso-
lution velocity. The literature has reported a higher dissolution
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

11. REVIEW 11
Figure 2. In vitro surface dissolution, (A) surface image dissolution in correspondence with diffusional layer thickness (h), and (B) intrinsic
dissolution profile in correspondence with the surface image dissolution.
rate for the nanosuspensions,66,67
which dissolve instanta-
neously as compared with micronized suspension. Hence, sink
condition dissolution is an appropriate tool for QC analysis.
However, because of the instantaneous dissolution behavior of
nanocrystals during sink condition, it is challenging to quan-
titatively discriminate dissolution rate enhancement with re-
spect to particle size. In addition, the long sampling interval
during conventional dissolution also adds to the difficulty. In
response to these problems, few scientists have tried alterna-
tive approaches.
Flow-Through Cell
The flow-through cell dissolution apparatus demonstrated the
capability of studying the dissolution of all kinds of formula-
tions, such as tablets, capsules, and powders.68,69
Heng et al.70
have carried out dissolution studies and compared the dissolu-
tion rate by asking the question, “What is the suitable disso-
lution method for drug nanoparticles?” Initial dissolution rates
were determined for the paddle, basket, and flow-through ap-
paratus. The initial dissolution rate was calculated from the
following equation:
dM
dt
Nanoparticles
dM
dt
Unprocessed
=
slope %dissolved
time
nanoparticles
slope %dissolved
Time
unprocessed
The paddle and basket dissolution apparatus were described
as inappropriate for nanoparticles because of their tendency to
form aggregates. The ratios obtained for the initial dissolution
rate enhancement in both cases did not agree with the model
predicted values. Dissolution studies via the dialysis process
(membrane with MWCO 12–14 kDa) were found to be very slow
and having a limited ability for size discrimination. As a result,
there was no significant difference noted in the dissolution pro-
files between nanoparticles and the unprocessed powder. Based
on the analysis, the flow-through cell set-up was described as
appropriate for dissolution of nanoparticulate powder by mini-
mizing the wetting problems.71
The experimental data for the
dissolution profiles were able to discriminate well with the un-
processed powder. The initial reported dissolution rate ratio
was the average number of the triplicate study and was in
agreement (6.95 vs. 7.97) with model predicted value. The au-
thors concluded it as the most suitable analytical process for
the dissolution of nanoparticulate powders. However, selection
of an appropriate flow rate is required for proper discrimination
because a change in the flow rate can generate change in the
release behavior. Note that the presence of air can also affect
the flow rate during the study, which may alter the dissolu-
tion behavior. Therefore, the study should be carried out with
caution.
Alternative Approaches
Nanosuspensions/nanocrystals exhibit instantaneous dissolu-
tion behavior, making it difficult to monitor them with conven-
tional methods, which requires sample withdrawals, as well as
dilution and filtration steps before quantitative analysis. In situ
fiber optic probes are not able to provide an accurate quantita-
tive determination because of the generation of absorbance by
both the dissolved and undissolved nanocrystalline particles.32
Recently, Kayaert et al.72
reported an alternative approach by
studying the dissolution behavior of nanosuspensions using a
solution calorimeter. The use of a solution calorimeter for dis-
solution study has been previously reported for polymer and
polymeric blends.73
This dissolution study was examined based
on appraising the change in the heat throughout the process. In
other words, heat required for the dissolution process is mea-
sured. The methodology consists of sealing the breakable glass
ampoule along with nanosuspension using bees wax. The glass
vial should be kept in the sample cell, surrounded by the me-
dia in which the dissolution study is intended to perform. After
reaching the required temperature, the glass vial should be
broken, and the dissolution process is started. The rate of dis-
solution can be then assessed by converting the raw data (i.e.,
temperature vs. time and cumulative heat vs. time) to percent-
age dissolved versus time. The dissolution process for nanosus-
pensions was observed to be extremely fast. It takes just 20 s to
complete the dissolution process for nanosuspension, whereas
in the case of crude suspension, it was found to be between
8 and 15 min For the traditional dissolution apparatus (i.e.,
basket and paddle), the earliest sample one can withdraw is
at 2 min. Hence, the solution calorimeter unquestionably pro-
vides the advantage of real time measurement compared with
traditional methods. Consumption and/or production of heat
can be monitored from the beginning of the dissolution process
without disturbing (i.e., sample withdrawal) the system. There
are drawbacks with this method, which need to be considered
for an accurate evaluation of the data generated. The method
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

12. 12 REVIEW
measures the summation of the total heat change, which in-
cludes contributions from the breakage of the glass ampoule,
heat change from other excipients in the formulations, and heat
change due to the dissolution process. Therefore, a careful eval-
uation of the data is warranted by considering all these con-
tributing factors. In addition, the reported dissolution time is
far shorter (i.e., 20 s to 15 min) using this method, but a single
experiment requires additional time for the equilibration before
the test and also requires post-test time after the dissolution,
making it more time consuming than the conventional dissolu-
tion methods. Furthermore, the instrument is very subtle and
requires a skilled operator. Recently, another separation-based
approach was reported by Shah et al.74
using a modified dial-
ysis method known as pulsatile microdialysis (PMD)75
for the
characterization of supersaturation and precipitation behavior
of poorly soluble drugs. Here, the pore size for the dialysis probe
was approximately 18 kDA (ß2 nm). Hence, the final sample
should contain only the dissolved drug. This analytical method
can be useful for characterization of high energy solid forms
(amorphous) or nanoamorphous33
forms because of the advan-
tage of quick sampling (i.e., 10 s). As a more convenient direct
sampling method, PMD may not be much useful for the study of
nanocrystals with the size range of 150–400 nm because of the
availability of the syringe filters with pore size 0.1 :m. The use
of 0.02 :m (ß20 nm) anotop syringe filters for the dissolution
study has also been reported in the literature.45
As a result, it
may be of great interest if the comparison of the sample anal-
ysis from PMD and the sample analysis from 0.02 :m syringe
filters has been carried out to describe the usefulness of the
method more precisely.
Another separation-based approach has been reported by
Murdande et al.51
by modifying the traditional sink dissolu-
tion to various non-sink conditions. Non-sink conditions can
be generated by pre-dissolving API in the dissolution me-
dia prior to perform the dissolution experiment. Recently, an-
other literature has also reported the dissolution of nanocrys-
talline suspension for poorly soluble molecules using non-sink
conditions.76
Because of the lack of discrimination during the
initial phase of dissolution by sink dissolution, the goal was
to slow down the dissolution velocity of nanocrystals and mi-
cronized crystals in such way that the initial dissolution phase
becomes more discriminative. Here, the samples were filtered
with both 0.02 :m (for nano) and 0.1 :m (for micro) after es-
tablishing the filter binding capacity of the each model com-
pounds. As the saturation level increases, the rate of disso-
lution decreases, creating a more discriminating portrayal of
the initial dissolution rate enhancement (i.e., at 75% satura-
tion level the initial rate of dissolution, 10 :m:362 nm:122 nm
were 1.0:1.8:3.6). The observed data look more promising com-
pared with the data obtained under sink conditions. However,
filtration from 20 nm pore size of filters should be performed
carefully because of the chance of blockage or clogging of the
pores by larger size particles. From our knowledge, all these
approaches are reported specifically for nanocrystals. One can
select any analytical method listed above based on their re-
quirements and the availability of resources.
PK BEHAVIOR OF CRYSTALLINE NANOPARTICLES
Different formulations have different effects on the PK profile
of the same drug. Besides chemical properties, a change in the
physical property (i.e., particle size reduction) may also have a
considerable impact on altering the PK behavior of a molecule.
The reduction of the particle size increases the dissolution ve-
locity and may have some effect on in vitro solubility. Similar
behavior was observed in the case of in vivo parameters, such
as AUC (+ve), Cmax (+ve), Tmax (−ve), and fed/fast variability
(−ve). A reduction of particle size improves the dissolution rate,
which increases the absorption of the drug in the body, and it
leads to an increase in PK performance. Compounds belong-
ing to the BCS Class II have issues related to poor solubility,
dissolution-rate-limited absorption, and bioavailability. Parti-
cle size reduction can improve the bioavailability of such com-
pounds by improving the dissolution rate. For the nanocrystals,
an increase in the oral bioavailability of the compounds can be
attributed to an increase in the SA.77
Improvement in Oral Bioavailability
An improvement in the dissolution rate and adhesion to the
gut wall can be considered the main contributing factors for
enhancing bioavailability and overall PK performance. After
oral administration, the formulation disintegrates and begins
the dissolution process. The dissolution rate resembles the
concentration gradient in the physiological environment. As
a result, an improvement in the dissolution rate leads to an
enhancement in the absorption, and finally, in the bioavail-
ability of the compound. Because of the smaller size of the
nanoparticles, they tend to adhere to the gut wall.78
Bioavail-
ability enhancement can be achieved both actively and pas-
sively. The classic example for bioavailability enhancement has
been represented in the case of danazol, which is poorly sol-
uble (<1 :g/mL) and one of the most challenging compound
to work within the pharmaceutical industry.79
In vivo studies
of nanosuspensions of danazol (169 nm) in beagle dogs have
shown enormous enhancement in Cmax and a 16-fold enhance-
ment in relative bioavailability, compared with the micronized
suspension.12
Therefore, it is understood that by reducing the
particle size, the solubility issue associated with danazol can
be solved. Moreover, it can also be delivered at a lower dose by
enhancing the therapeutic outcome of the compound. FBT is
another classic example of a poorly soluble drug with a high log
P (i.e., 5.3) value. FBT is used in hypercholesterolemia and has
a solubility and dissolution-rate-limited absorption property.
Bioavailability of FBT is attributed to the dissolution behavior
of particles in the gastric environment. A comparison of the PK
profile of nanocrystals and coarse powder demonstrated signif-
icant enhancement in the rate of absorption after oral adminis-
tration. The Cmax of nanocrystals was observed to be five times
higher than that of the coarse powder, along with a higher AUC
and Tmax.80
Zuo et al.40
made an effort to compare formulated
nanocrystalline particles with the commercial nanocrystalline
formulation LipidilTM
-ez with respect to in vitro and in vivo
behavior. No significant difference was observed between the
two nanocrystalline formulations, suggesting that variability
will be less when particle size is reduced to nanometer range.
Certainly, the re-dispersion behavior of dried nanocrystals also
plays a contributing role by demonstrating reversible or irre-
versible aggregation behavior. Bioavailability can be altered by
several factors in the gastric environment, such as pH change,
food effect, the presence of salts, and so on. In all the reported
cases reported in the Table 4,12,40,80–88
a significant enhance-
ment in dissolution rate was observed for the nanocrystals,
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

13. REVIEW 13
Table 4. Effect on In Vivo Parameters
Compound Animal Model PK Parameters Comment Reference
Aripiprazole Beagle dogs NSa (350 nm) Coarse suspension 71% enhancement in
relative bioavailability
81
Tmax (h) 1.04 ± 0.24 3.33 ± 1.50
Cmax (ng/mL) 137.37 ± 17.38 43.10 ± 11.68
AUC (ng h/mL) 618.15 ± 81.28 386.06 ± 78.54
Nitrendipine Rat NS (209 nm) Tablet Fivefold enhancement in
relative bioavailability
82
Tmax (h) 1 ± 0 2.2 ± 1.17
Cmax (:g/mL) 3.65 ± 0.43 0.60 ± 0.11
AUC (:g h/mL) 17.12 ± 2.59 3.44 ± 0.47
Danazol Beagle dogs NS (169 nm) Coarse suspension 16-fold enhancement in
relative bioavailability
12
Tmax (h) 1.5 ± 0.3 1.7 ± 0.4
Cmax (:g/mL) 3.01 ± 0.80 0.20 ± 0.06
AUC (:g h/mL) 16.5 ± 3.2 1.0 ± 0.4
Naproxen Rat NS (270 nm) Unmilled suspension ß1.25-fold enhancement in
relative bioavailability
83
Tmax (min) 23.7 ± 5.1 33.5 ± 2.9
Cmax (:g/mL) 187 ± 18 126 ± 4
AUC (:g min/mL) 19,062 ± 573 15228 ± 994
Apigenin Rat NCb (400—800 nm) Coarse powder ß3.5-fold enhancement in
relative bioavailability
84
Tmax (min) 90 ± 14 120 ± 16
Cmax (:g/mL) 5.4 ± 0.6 1.5 ± 0.2
AUC (:g min/mL) 1509 ± 196 445 ± 45
Cefpodoxime proxetil Rabbit SDNSc (<300 nm) Microsuspension ß1.6-fold enhancement in
relative bioavailability
85
Tmax (h) 0.75 ± 0.11 1.75 ± 0.68
Cmax (:g/mL) 18.36 ± 2.03 10.88 ± 1.01
AUC (mg h/mL) 47.55 ± 4.33 29.78 ± 3.47
Baicalein Rat Baicalin NC (335 nm) Coarse powder ß1.6-fold enhancement in
relative bioavailability
86
Tmax (h) 0.92 ± 0.38 1.67 ± 0.29
Cmax (:g/mL) 11.12 ± 1.25 7.18 ± 1.25
AUC (:g h/mL) 119.25 ± 20.26 71.41 ± 4.38
Simvastatin Rat NC (387 nm) Coarse powder ß1.5-fold enhancement in
relative bioavailability
87
Tmax (h) 1.99 ± 0.05 2.88 ± 0.08
Cmax (ng/mL) 450.3 ± 140.5 300.2 ± 67.01
AUC (ng h/mL) 1110.3 ± 280.62 770.9 ± 110.3
Fenofibrate Rabbit NC (460 nm) Coarse powder ß4.7-fold enhancement in
relative bioavailability
80
Tmax (h) ß0.4 ß1
Cmax (:g/mL) 536.66 ± 35.09 113.46 ± 29.05
AUC (:g h/mL) 134.38 ± 6.47 28.41 ± 5.52
BMS-347070 Beagle dog NC Micronized ß2.7-fold enhancement in
relative bioavailability
88
Tmax (h) 2 3
Cmax (ng/mL) 1475 ± 375 483 ± 79
AUC (ng h/mL) 28,613 ± 3850 10,870 ± 1651
Fenofibrate Beagle dog SDNCd LipidilTM-ez No significant difference in
relative bioavailability
40
Tmax (h) 2.8 ± 1.8 5.2 ± 9.2
Cmax (ng/mL) 2075.2 ± 1101.1 2349.5 ± 1050.5
AUC (ng h/mL) 30,496 ± 6541 34,035.9 ± 19,286.4
a
NS, nanosuspension.
b
NC, nanocrystal.
c
SDNS, spray-dried nanosuspension.
d
SDNC, spray-dried nanocrystals.
which caused a significant reduction in the time to reach max-
imum concentration, an improvement in the rate of absorp-
tion, peak plasma concentration, and enhancement in relative
bioavailability.
Food Effect
In most cases, food increases the bioavailability of the drug by
increasing bile secretion and increasing the duration of gas-
tric emptying time. For a drug molecule’s clinical efficacy and
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

14. 14 REVIEW
Table 5. Food Effect
PK Parameter
Fed Fast
Compound NSa Refb NS Ref Reference
ELND-006 Tmax (h) 1.4 3 1.4 1.8 90
Cmax (ng/mL) 365 159 294 49.4
AUC (ng h/mL) 3063 1767 2430 315
Fc 110 63.4 87.2 11.3
Fed Fast
NCd Jetmilled NC Jetmilled
Cilostazole Tmax (h) 1 1 1.3 ± 0.5 1 91
Cmax (ng/mL) 4872 ± 112 2901 ± 314 5371 ± 1173 1029 ± 218
AUC (ng h/mL) 13,589 ± 3895 10,669 ± 3417 17,832 ± 4994 2875 ± 587
F 0.67 ± 0.22 0.53 ± 0.21 0.86 ± 0.29 0.15 ± 0.04
Fed Fast
SNCDe SDDf SNCD SDD
Ziprasidone Cmax (ng/mL) 260 285 416 140 92
AUC (ng h/mL) 1911 1949 2044 879
a
NS, nanosuspensions.
b
Ref, reference sample.
c
Relative bioavailability.
d
NC, nanocrystal.
e
SNCD, solid nanocrystalline dispersion.
f
SDD, solid amorphous spray-dried dispersion.
future success, persistence in oral bioavailability can only be
achieved by eliminating the food effect.89
The presence of bile
salts in the gastric environment also has an impact on the dis-
solution behavior of the molecule. It has been reported that the
presence of food reduces the dissolution behavior of micronized
particles in certain cases as reported in Table 5.90–92
The ratio
of bioavailability of the fed to fast state was observed to be close
to 1 in the case of nanocrystals, while micronized formulation
of the same drug showed a significantly higher ratio (approx.
sixfold) of the fed to fast state by demonstrating the poten-
tial food effect after oral administration to an animal model
with the same dosing. Bile salt secretion usually increases
in the fed state.93
A change in concentration of the bile salt
in the stomach leads to a change in dissolution behavior and
slows down or enhances absorption of the drug as depicted in
Figure 3. This phenomenon causes a large variability from pa-
tient to patient and also from fed state to fast state. Micronized
or larger particles that have shown improved absorption in the
fed state might be because of the micelle formation. For the
nanocrystals though, they are not helping in improving the in-
trinsic solubility, but they provide an advantage by enhancing
the initial dissolution rate because of the larger SA. The higher
rate of dissolution leads to an increased rate of absorption, and
eventually, enhancement in the overall bioavailability, irrespec-
tive of the fed or fast state. Hence, it can be stated that the bile
salts concentrations have significantly less effect on the absorp-
tion behavior of nanoparticles. The same observation has been
reported for cilostazole91
[fed/fast ratio—0.78 (nano vs. 3.53
jet-milled)]. Thombre et al.92
have reported the PK behavior of
an anti-psychotic drug ziprasidone in beagle dogs by conduct-
ing the fed- and fasted-state bioavailability experiment for the
both solid nanocrystalline dispersion and the commercial blend
(amorphous spray dried dispersion). The commercial blend has
shown a twofold improvement in bioavailability in the fed state
as compared with the fasted state, indicating the failure of the
commercial blend to prevent variability in absorption behavior
irrespective of the food state. A similar food effect was observed
with human data. On the other hand, nanocrystals have proven
to be efficient enough to prevent the variability from fed to fast
state by generating a similar bioavailability profile in beagle
dogs. From the reported literature, it can be concluded that vul-
nerability in the variation of oral bioavailability for potential
drugs can be avoided with reduction in particle size to nanome-
ter scale.
Particle Size Effect: Micro Versus Nano
The effect of particle size on in vitro and in vivo performance is
well documented elsewhere.91,94–96
Different particle sizes have
a different effect on the initial dissolution rate, the rate of ab-
sorption, and on oral bioavailability. Moreover, particles with
nanometer size range may have the tendency to pass through
both active and passive diffusion pathway, which increases the
chances for sub-micron particles to reach to peak plasma con-
centration with a reduction in time compared to macro/large
particles. Sun et al.97
have shown the effects of particle size,
ranging from micron to nanometer size, on Cmax, Tmax, and
AUC.98
A large increase in bioavailability was observed for the
both nanoparticles (i.e., 300 and 750 nm) as compared with
the micron size particles and coarse powder, which is reported
in Table 6. The rise in bioavailability can be attributed to in-
creased SA and adhesion behavior of nanosized particles. There
Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694

15. REVIEW 15
Figure 3. Effect of food on dissolution of nanocrystals and microcrystals.
Table 6. Particle Size Effect: Micro versus Nano
PK Parameter
Compound Size (nm) Tmax (h) Cmax (ng/mL) AUC (:g h/mL) Reference
Itraconazole 300 3 712 ± 121 9967 ± 2527 97
750 4 501 ± 73 8649 ± 1580
5500 3 218 ± 86 1271 ± 398
Powder – 40 ± 9 197 ± 61
Nitrendipine 200 1.0 ± 0.0 3.65 ± 0.43 17.12 ± 2.59 98
620 1.0 ± 0.0 2.75 ± 0.50 14.36 ± 2.75
2700 0.8 ± 0.1 1.74 ± 0.26 8.19 ± 0.85
4100 0.7 ± 0.1 1.19 ± 0.08 7.41 ± 0.46
20,200 3.4 ± 1.2 1.04 ± 0.21 6.89 ± 1.15
Powder 4.1 ± 2.4 0.42 ± 0.08 2.76 ± 0.46
was significant difference observed in the dissolution profiles
between both of the nanoparticles (i.e., 300 and 750 nm), but
no significant difference has been reported in the PK profiles.
The same observation was reported by Xia et al.98
by studying
effect of particle size on the oral bioavailability of nitrendipine
in the rat model. The overall bioavailability was reported to
be a ninefold increase compared with the coarse powder and a
threefold increase as compared with the micronized particles.
Comparing the PK parameters for both nanoparticles (200 vs.
620 nm), a slight improvement was noted in Cmax, Tmax, and
AUC. Based on the literature data, an interesting area for a
further research is possible, by lowering the particle size down
to the 50 nm range with a narrow size distribution and mea-
suring the relative bioavailability. This may have more pro-
nounced effect on the bioavailability because of the further im-
provement in the thermodynamic solubility suggested by the
Ostwald–Freundlich equation. A comparison of adhesion rates,
along with the various particle size ranges, starting from 50 nm
to micron level, will also be an interesting area to explore for
further understanding the particle size effect on the bioavail-
ability.
SUMMARY
Drug nanocrystal formulation is a well-established and suc-
cessful approach, and it is described in the initial phase of
this review article. Nanocrystal formulation approach is more
promising because most of the major pharmaceutical compa-
nies have adopted this approach because of its effectiveness and
convenience. During the early phase development and during
formulation development for pre-clinical studies, it is more con-
venient to formulate the nanocrystal for the determination of
the exposure and the PK parameters to confirm the candidacy
DOI 10.1002/jps.24694 Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

16. 16 REVIEW
of the drug molecule. It is also important to know the thermody-
namic and the kinetic behavior with respect to size reduction.
Several approaches have been tried by researchers for the accu-
rate determination of thermodynamic solubility (Tables 2 and
3). Some have reported suspicious results due to generation of
artifacts by the analytical method for the solubility determi-
nation. Despite the shortcomings of some of the conventional
methods for determining solubility (specifically for nanoparti-
cles), others have reported excellent results by adopting newer
approaches for the solubility determination. The boost in ther-
modynamic solubility can be observed only when the particle
size is reduced to approximately 50–80 nm, in the remainder
of the cases, only kinetic boost (i.e., the dissolution rate) can
be observed without greatly impacting the thermodynamic sol-
ubility. As the nanocrystals have a high dissolution velocity,
the traditional dissolution method is not suitable to discrimi-
nate the quantitative difference in the dissolution behavior for
the nanoparticle. However, there are several alternative ap-
proaches that have been described in this review article for
discriminative analysis of the dissolution rate enhancement
upon nanosizing. The initial dissolution rate enhancement as-
sociated with particle size is a potential parameter for the esti-
mation of bioavailability enhancement. More advanced work is
warranted in this area to understand the behavior of nanocrys-
tals and to avoid excessive animal studies. The effect of food is
found to be negligible in the case of nanocrystals as compared
with microcrystals. The particle size is shown to be a dominant
characteristic in governing dissolution and bioavailability per-
formances. Furthermore, some issues such as rate of adhesion
along with particle size change, and the mechanism of diffusion
(i.e., active or passive) across the cell membrane still require
additional study. The field of nanocrystal is growing rapidly
and is receiving the attention of researchers. In the coming
decade, nano formulations have great potential to go beyond
its present level, largely because of having the advantage of
delivering large and small drug molecules at specific targets.
Conflict of interest: The authors reports no conflict of interest.
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Shah, Murdande, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24694