Journal of Pharmaceutical Sciences 109 (2020) 3235-3247

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences

journal homepage: www.jpharmsci.org

Review

Freeze-Drying of Pharmaceutical and Nutraceutical Nanoparticles:

The Effects of Formulation and Technique Parameters on
Nanoparticles Characteristics
Mohsen Mohammady a, Yasaman mohammadi a, Gholamhossein Yousefi a, b, *
a
Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran
b
Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Nanoparticles (NPs) are of the most interesting novel vehicles for effective drug delivery to humans.
Received 6 April 2020 Freeze drying is known as an engaging process to improve the long lasting stability of NPs formulations.
Revised 23 May 2020 This study aims to elucidate the importance of various parameters involving in freeze-drying of the most
Accepted 15 July 2020
common pharmaceutical/nutraceutical NPs including nanosuspensions, nanocrystals (NCs), cocrystals/
Available online 20 July 2020
nanococrystals, nanoemulsions (NEs), nanocapsules (NCPs) and nanospheres (NSPs). Regarding this, the
therapeutic goals of NPs and specifications of drug must be considered. According to our survey, the most
Keywords:
Nanoparticles influential factors for achieving optimum results include type and concentration of cryoprotectant/lyo-
Lyophilization protectant, stabilizer structure and concentration, the NPs concentration in solution, freezing, annealing,
Nanocrystal and drying rate, the interaction between protectants and stabilizer, solvent type and antisolvent to
Nanococrystals
solvent ratio. The study shows that for each class of NPs, specific variables are of highest significance and
Nanoemulsion
Nanospheres should be optimized. For instance, about NCs, freezing rate and antisolvent/solvent ratio should be
Nanosuspension particularly considered and for emulsified NPs, the best results have been obtained by 5e20% of sac-
charides as cryoprotectants. These findings suggest that to obtain a product with the lowest aggregation
and particle size (PS), optimization of the effective factors in formulation and lyophilization process are
essential.
®
© 2020 American Pharmacists Association . Published by Elsevier Inc. All rights reserved.

Introduction improve drug permeation through biological barriers for instance,

Nanoparticles
The drug delivery to humans based on NPs is of the most
interesting strategies recently employed for achieving more effec-
tive drug therapy with lower side effects in various administration
routes.1 Generally, the word “NP” refers to particles with sizes in the
range of 1e100 nm. However, in the field of pharmaceuticals and
medicine, submicron particles are normally called NPs.2,3 The NPs
give numerous profits over microparticles. The submicron size of
NPs causes to higher intracellular uptake compared to micropar-
ticles.4 Routinely, due to their capability to keep the loaded drug
stable and provide it in a consistent manner, NPs can significantly
enhance the bioavailability of many drugs. Moreover, NPs can
* Corresponding author. The Department of Pharmaceutics, School of Pharmacy,
Shiraz University of Medical Sciences, Shiraz, Iran.
E-mail address: ghyousefi@sums.ac.ir (G. Yousefi).
intestinal and respiratory epithelia.5,6 NPs are mostly fabricated in
aqueous media. However, these aqueous dispersions are not actu-
ally stable making them susceptible to aggregation and fusion of
particles throughout long periods of storage.7 Additionally, the
other physical stability difficulties are common with NPs including
sedimentation, creaming (the tendency of oil droplets for aggre-
gating to diminish the surface energy), agglomeration, crystal
growth (Ostwald ripening) and change of crystallinity state
(changing from amorphous to crystalline).8 The chemical instability
of NPs structure includes the hydrolysis of polymeric materials
which depends on the conditions in which the NPs are stored such
as temperature, medium pH and polymer characteristics (for
instance its structure and molecular weight). This kind of instability
is frequently reported during NPs storage for long time.9,10 There-
fore, when developing the NPs formulations, how physically and
chemically stable they remain in long periods of time can be a
detrimental issue.11
Freeze drying is a routinely used industrial process to improve
the long-term chemical stability of pharmaceutical formulations

https://doi.org/10.1016/j.xphs.2020.07.015
0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
3236 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
including NPs owing to its unique advantages. Particularly, its
possibility of application for small volume products and its rather
simple scale up are of high significance.12 Freeze-drying is by
definition a low temperature dehydration process by transition
from the solid to the gas state without passing through a liquid
state. It consists of three sequential steps of freezing, primary
drying and secondary drying.13 Through the process of sublimation,
water is eventually eliminated from ice at pressures and tempera-
tures below the triple point of water.14 Lyophilized formulations
give straightforward handling during shipping and storage.15
Similar to solid state medicines like pills or capsules, the NPs as
liquid dispersions should be firstly converted to dry powders and
next converted to other solid dosage forms if needed.16 In the
course of the procedure, freezing and dehydration stresses are
developed and they might probably enhance the aggregation of NPs
causing to instability. Different excipients like cryoprotectants and
lyoprotectants could also be utilized to decrease the freeze-drying
stresses, and conserve the physicochemical characteristics of
NPs.17 This study aims to obtain an updated insight into the es-
sentials involving in the lyophilization of pharmaceutical and nu-
traceutical NPs including nanosuspensions, NCs, NEs, NCPs and
NSPs. Mainly, it focuses on the formulation considerations and
freeze-drying cycle optimizations to obtain a suitable lyophilized
product.
Freeze Drying Stages
The procedure of freeze drying encompasses three essential
steps. First one is freezing or solidification, next is primary drying,
also called ice sublimation and finally, secondary drying which is
moisture desorption. Primarily in the process of freeze-drying, the
liquid suspension is frozen and consequently, the pure water con-
verts into ice crystals. As the process continues, the proportion of
ice crystals and the concentration of the still existing liquid in-
crease. Consequently, the viscosity of product increases and this
results in inducing restraint for further crystallization. As this vi-
cious and concentrated liquid becomes solid, an amorphous, crys-
talline, or combined amorphous-crystalline solid will be obtained.18
“Bound water” is the small amount of water which does not freeze
and remains liquid. Drying step is the process which the ice from
the frozen product is sublimated.19 At the first stage, heat is con-
ducted from the shelf through the tray and subsequently to the
frozen solution and directed to the subliming front. At the second
stage, the sublimed ice changes into vapor and goes through the
dehydrated part to the top of the sample. At the third stage, the
water vapor is moved through the chamber to the condenser. At the
final stage, the water vapor condenses and a permeable plug is
shaped in which small openings can be seen in the place of ice
crystals.20 At secondary drying step, the water which is not isolated
as ice nor sublimated off is removed from the product.21 Fig. 1
depicts the above-mentioned stages of freeze drying process
regarding the how of water removal from NPs suspensions.
Cryoprotectants and Lyoprotectants
Different stresses are developed throughout freeze-drying pro-
cess. These stresses result into worsening of the texture of the mass
due to the shock caused by low temperature, osmotic shrinkage and
particles aggregation when rehydrating.22 Within the cryo-
concentrated phase, high concentration of NPs might induce their
aggregation or fusion. Besides, NPs might lose their stability as a
result of the mechanical pressures produced by ice crystals. To
prevent the mentioned events, several excipients are also included
into the NPs formulation before freeze-drying.17 In this case, the
excipients are inert substances added to a pharmaceutical product
to boost stability during lyophilization and also improve physical
stability of the product at storage time.23 Excipients used for this
purpose are divided into two categories of cryoprotectant and
lyoprotectant agents. Cryoprotectants shield the product through
the freezing step and lyoprotectants prevent from the stresses
caused during the drying step. The protecting impact of cryopro-
tectants is described by many elements. As freezing is applied in a
temperature lower than glass transition temperature (Tg) of cryo-
protectant, immobility of particles in its glassy matrix increases the
stability of the product.24 Various approaches which protect the
NPs native structure, rely on replacing the water. This hypothesis
might explain the NPs stabilization by hydrogen bonds between the
polar groups at the surface of NPs and stabilizer molecules caused
by water losing. Therefore, the characteristics of NPs are saved by
the stabilizers through acting as water replacement. Besides, add-
ing cryoprotectant agents that increase the viscosity within the
sample can subdue the construction of ice crystals. Consequently,
aggregation through the freezing step in temperatures above Tg is
averted by the isolation of separate particles.25 The most typical
cryoprotectants are sugars including monosaccharides (glucose,
fructose, and galactose), dissaccharides (sucrose, lactose, trehalose,
and maltose) and tri-saccharides (Raffinose).
Trehalose, due to lack of internal hydrogen bonding, low hy-
groscopicity, low chemical reactivity and high Tg, is more efficient
than other sugars.26 For stabilizing the PS of chitosan NPs, glucose,
sucrose, trehalose, mannitol, polyethylene glycol (PEG) 2000, and
PEG 10000 cryoprotectants at concentrations of 5%, 10%, 20% and
50% were employed. Among these cryoprotectants, the best pro-
tection was observed for sucrose and trehalose at concentration of
5% such that there was no growth in the PS due to the lack of ag-
gregation. Other cryoprotectants had little effect and there was a
significant rise in the size of the particles. For instance, when PEG
10000 was used as cryoprotectant, particles enlarged significantly
in the samples.27 On the other hand, destabilization of NPs by a
typical cryoprotectant has been reported by some researchers. In
the case of increase in the concentration of a cryoprotectant in NPs
formulation, both raise or reduction in NPs’ aggregation can be
observed.28
The protective effect of polyols (glycerol, mannitol, sorbitol,
adonitol, and inositol) has been in general lower than that observed
for sugars. Mannitol due to its potential for crystallization and also
forming eutectic mixture with ice will separate the phases within
the cryo-concentrated portion of the frozen NPs without any stable
interactions with them. Separate NPs in the NP-rich phase will
interplay and form aggregates. Furthermore, crystals of water and
mannitol which are enlarged may apply mechanical stresses on the
NPs leading to their fusion. Therefore, a minimum number of
mannitol molecules should stay molecularly scattered in the
amorphous NPs phase in a stabilization process.29 X-ray diffraction
discovered that monosaccharide-including freeze-dried
griseofulvin-lipids NPs had crystalline structure. Quite the oppo-
site, disaccharide-including freeze-dried griseofulvin-lipids NPs
showed an amorphous structure.30 Polymers (dextran, starch, PEG,
and polyvinyl pyrrolidone) have stabilizing role in NPs. PEGs have
additionally been utilized as stabilizer in freeze drying of poly-
electrolyte complex (PEC) NPs as well as chitosan NPs. A rise in
trehalose concentration up to 2% w/v caused a reduction in the PS
ratio (defined as the ratio of PS after to before freeze-drying) and
slight PEC NPs aggregation. When PEC NPs were freeze dried with
3, 4 or 5% w/v of trehalose, PS ratio decreased and aggregation was
not seen. With 5% w/v trehalose not reduction in PS neither ag-
gregation of particles were seen, thus at 5% w/v, trehalose acted in
the role of an efficient cryoprotectant for PEC NPs. Also, the use of
polymer PEG could be more effective than trehalose so that 1% of
PEG 2KD in contrast to 3% of trehalose could make redispersibility
 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
acceptable. Best protection for NPs was obtained by using 2% PEG
which was comparable to 4% trehalose. The combination of treha-
lose and PEG 2KD as protectant produced a synergistic effect
reducing the PS ratio and the aggregation of NPs. Therefore, it can
be concluded that adding a polymer like PEG can reduce the con-
centration of the sugar required for lyophilization.31 Therefore, the
use of suitable cryoprotectant at optimal concentration results in
significant positive effects in controlling the distribution of PS and
shape. The application of trehalose, mannitol, sorbitol or glycerol as
cryoprotectant is a useful method for freeze-drying of NPs to keep
up their physical characteristics.32 Consequently, cryo and lyopro-
tectant type and concentration, protectant to NPs mass ratio and
the possibility of crystal or amorph formation are important vari-
ables in protectant selection for stabilizing NPs. Fig. 2 demonstrates
the role of cryoprotectants in prevention of NPs aggregation after
rehydration of lyophilized NPs.
Freeze Drying of Pharmaceutical NPs
Nanosuspensions
The nanosuspensions have extended the innovative and assur-
ing capacities for delivery of water-insoluble drugs.33
Fig. 1. The varying stages in freeze drying of nanoparticles and the phase transitions of product water through different stages of the process.
3237
Nanosuspensions, described as colloidal dispersions of nano-sized
medicine particles, may attain great drug loading efficiency (up to
100%) in drug delivery systems.16 Normally, nanosuspensions made
stable by surfactants or polymeric steric stabilizers and constructed
by appropriate techniques may cause to a smart stability.34 But, the
most of NPs are chemically and physically less stable in aqueous
suspensions. NPs are often formed in an aqueous environment and
this environment causes to physical instability as particle aggre-
gation and fusion over time.7 NP suspensions are also susceptible to
the growth of microorganisms.8 To solve this problem, a suspension
containing a NP should be formulated as a solid. Lyophilization is an
answer to this problem with the aid of various excipients such as
cryoprotectants and lyoprotectants which are used to reduce
lyophilization stress and maintain physical and chemical proper-
ties.28 As a whole, lyophilized NPs are considerably aggregative and
particle dimentions rise once it is reconstituted into solution
despite of having surfactants in their structure.35 The use of sugars
due to the effect on Tg has an important role in their lyophiliza-
tion.36 It is declared that poly (DL-lactide) -poly(ethylene oxide)
(PLA-PEO) NPs have been aggregated during freeze-drying and the
complication as a major drawback could be bypassed by using
trehalose in the role of a cryoprotectant.37 Optimization of lyoph-
ilization includes the selection of suitable cryoprotectant, freezing
3238 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
Fig. 2. The role of protecting agents in prevention of nanoparticles aggregation after reconstitution of lyophilized nanoparticles.
rate, formulation characteristics, and lyophilization cycle.38
Therefore, decent lyophilization of NPs results in the easy re-
suspending of NPs, the minimum change in particle dimensions
and distribution, and maintaining the activity of the loaded drug.20
Table 1 summarizes the freeze-dried nanosuspensions found in
literature including the manufacturing method and main excipi-
ents used to optimize freeze-drying.
Formulation Effect
The formulation parameters including polymer structure and
concentration, type of the surfactant and its concentration, and the
nature and concentration of cryoprotectant and lyoprotectant in
lyophilization are important.38 In order to increase the stability of
NPs and minimize their aggregation, polymers are routinely used.
Throughout the lyophilization process, these polymers might have
an impact on stabilizing NPs. It has been revealed that polyvinyl
alcohol (PVA) as a stabilizer can be the reason of small PSs.39 In
caprolactone NPs stabilized with 2.5e5% of PVA, no significant
aggregation was observed after lyophilization.38 This observation
was possibly the consequence of PVA being present on the surface
of the NPs which affects improvement of freezing.40 However, all of
the stabilizers do not increase the stability of NPs throughout
lyophilization. Additionally, the medicine trapped inside the NPs or
free unloaded drug are effective on the lyophilization process. If the
drug is free, it reduces zeta potential and leads to aggregation. For
example, lyophilization of cyclosporine NPs in the presence of
pluronic F68 was improved as a result of free drug adsorption onto
Table 1
The Examples of Drug Nanosuspensions Manufacturing Methods and Main Excipients Used for Freeze-Drying.
Compound Method
Naproxen Custom made apparatus (the yttrium-stabilized zirconium beads)
Azithromycin HPH
Carvedilol Anti-solvent precipitatione ultrasonication
Loviride Media milling
Silymarin Sonoprecipitation
Danazol Wet milling/HPH
Flurbiprofen High pressure homogenization
Loratadine Anti-solvent precipitation
Celecoxib HPH
the NPs surface.41 In another study, the importance of the combi-
nation of the two elements “steric stabilizer concentration” and
“cryoprotectant concentration” was disclosed to affect mutually
throughout the freezing phase.42,43 Increasing the concentration of
steric stabilizer lowered the propensity of particles aggregation.
Raising the steric stabilizer concentration generated a thicker steric
layer on the surface of the NPs that separated them within the cryo-
concentrated regions. The obtained results proved that while a rise
in the steric stabilizer concentration increased the stability of NPs, a
rise in the disaccharide concentration along with higher concen-
trations of steric stabilizer was notably more efficient in reducing
the size of particles for nanosuspensions.44
Cryo& Lyo Protectants Effect
Lyophilization may cause mechanical stress on nanosuspensions
which to solve this problem, the addition of some excipients as
cryoprotectant and lyoprotectant to the formulation is considered
as an option. Cryoprotectants prevent aggregation by protecting
NPs via the matrix mechanism and the hypothesis of separating the
particles in the unfrozen phase.21,25 The most commonly used
cryoprotectants are sugars with trehalose having more benefits
than other sugars. The stability of system depends on the concen-
tration and mass ratio of cryoprotectant to NPs. It has been argued
that PLA-PEO re-dispersion after lyophilization occurred at a 1:1
mass ratio of trehalose to NPs.37 Another study showed that,
resveratrol NPs with quercetin as stabilizer and 1% w/w of mannitol
and trehalose produced large NPs due to decreased cryoprotectant
Aid Excipients for Freeze-Drying Literature Reference
28
Lactose, sucrose, mannitol, PEG 2000
119
Poloxamer 188
120
Maltose
121
Sucrose
122
Mannitol
123
Mannitol
124
e
125
Sucrose
126
Lactose, sucrose, trehalose or mannitol
 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
concentrations. However, increasing the concentration of mannitol
and trehalose up to 5% w/w improved re-dispersion of resveratrol
NPs.45 Lyoprotectants are required to prevent drying stress by the
mechanism of forming hydrogen bonds between the lyoprotectants
and the polar groups of NPs' surfaces.29 NPs’ concentration in this
mechanism is important and it has been observed that a high
concentration of NPs resulted in high efficiency of lyoprotectants.46
Besides, it has been argued that re-dispersibility increases by
increasing the molecular weight of cryoprotectant. It was proved
that the aggregation of drug NPs can be avoided more efficiently
when PEG had a higher molecular weight. On the other hand, PEG
with the lowest molecular weight (PEG 0.4 k) led to an important
aggregation that was shown as particles which were bigger in size
relative to initial sizes.13
Freeze-Drying Cycle Effect
Freezing is the first step in the process of lyophilization, which is
defined by cooling the formulation and forming crystalline ice.
During freezing, the viscosity of concentrated residual suspension
increases and prevents crystallization.47 A solution, when reaches
to a temperature of Tg, has the highest concentration of cryo-
concentrated solution. Above Tg, concentration is not altered by
cooling, so the freezing temperature should be lower than Tg and be
adequate for a sufficient period to guarantee the perfect solidifying
of suspension.48 There is a controversy about the optimal slow or
fast freezing rate of NPs. Some studies have reported that fast
freezing has reduced PS compared to slow freezing.49 When pro-
tectant is added to the formulation, usually the freezing rate does
not affect the PS.50 It has been reported that fast freezing in the
presence of mannitol caused to a smaller PS than slow freezing.30 In
another study, slow freezing was more efficient for lyophilization
regarding size. For example, in the evaluation of the effect of
cryoprotectant on freezing rate of hydroxypropyl cellulose (HPC),
slow freezing was proved to be better.28 It was observed that with
varying the rate of freezing and the concentration of cryoprotectant
simultaneously, a faster rate of freezing and a higher concentration
were superior. However, when the freezing rate was relatively slow,
it caused to greatly decrease in the rate of NPs aggregation. The
slower rate of freezing may stimulate the migration of cryopro-
tectants to a cryo-concentrated liquid phase, leading to an increase
in its native concentration which subsequently prevents the NPs
aggregation.28 Moreover, the time of primary drying depends on
the depth of the vial and the formulation. The heat runs from the
lowest to the highest part and sublimation is conversely carried out
from highest to lowest part in the vial.19 Therefore, the process of
improving the lyophilization cycle focuses on shortening the pri-
mary drying period that is the longest stage.51
Nanocrystals
Drug NCs are in fact nanoscopic crystals of the parent drug
combined with excipients with sizes smaller than 1000 nm.52 The
Table 2
The Examples of Drug Nanocrystals Manufacturing Methods and Main Excipients Used for Freeze-Drying.
Compound Method
Albendazole HPH
Danazol Media milling
Naproxen Media milling
Oridonin HPH
Ascorbylpalmitate HPH
Fenofibrate Antisolvent percipitation
Ursodeoxycholic acid High-pressure precipitation tandem homogenization
Risperidone (Nanoprecipitation with probe sonication/
nanoprecipitation with HPH/nanoprecipitation
3239
processes to prepare NCs are often classified as top-down and
bottom-up. High-pressure homogenization53 and also wet ball
milling are considered as common top-down processes.54 Draw-
backs of the so called processes can be the utilization of surfactants,
the lengthy processing times, particle size polydispersity, low
yields, great energy consumption and potential air pollution com-
ing from the means of grinding.55e57 Bottom-up processes are
primarily precipitation ones. An important drawback of the most
commonly used bottom-up processes is the inability to properly
control the eventual size of drug crystals.57,58 In spite of their
benefits, drug NCs have numerous disadvantages such as complex
scaling up,55,58,59 possibility of nano-toxicities60 and stability
issues.61e63 One of the most crucial points to make sure these drug
products are safe and efficient is their chemical stability. Freeze-
drying and spray drying are among the most typical solidification
processes for NCs. Since a great deal of solid NCs are typically
reconstituted into nanosuspensions, their growing or agglomera-
tion must be avoided through drying process. This consideration
helps to keep up the nano-sized properties of NCs including fast
dissolution after the reconstitution. In order to cope with the sta-
bility problems during the solidification process, matrix formers
like mannitol, sucrose and polysaccharides can be added to NCs
formulations.62 Liquid NC dispersions designed for administration
as solid dosage forms should be freeze-dried to produce nano-sized
crystalline materials.64 An aqueous solution of cryoprotectant is
mixed with organic drug solution and after nano-precipitation of
crystals and organic solvent evaporation, the lyophilization take
place. The medicine chosen to be used in the technique ought to
have a low degree of glass transition (Tg). In order to reach to the
drug crystals, the freeze-drying temperature must be more than
drug's Tg.65 Robust agglomerates can be formed from NCs in the
case of unsuitable drying making their re-dispersion difficult. Thus,
it is of vital importance that the NPs remain discrete (re-dispers-
ible) throughout the drying step.66 Table 2 summarizes the freeze-
dried NCs found in literature including the manufacturing method
and main excipients used to optimize freeze-drying.
Lyophilization Process Effect
Polymers' steric stability becomes inactive during freeze-drying
of NCs dispersions. Through the extension of the freezing interface,
the major trap of polymer chains between NCs takes place.66 After
freezing, polymers and NCs merely have enough movement for
involvement or crystal fusion.67 Accordingly, the freezing step is of
much greater importance compared to sublimation step in order to
stop NPs from aggregating and save their ability to be re-dispersed.
Significantly, the pace of fronts freezing (freezing rate), is identified
primarily by gradient and may be a vital feature. Critical freezing
rate is described according to the plot of the particle dimensions of
rebuilt NCs against the freezing speed. The rate of critical freezing
relies upon the empirical PS measuring details depended on API
concentration.68 Quicker freezing rate yielded dry powders which
would be re-dispersed in water to produce the initial NPs.
Aid Excipients for Freeze-Drying Literature Reference
127
Mannitol
128
PVP, Tween® 80, mannitol, sucrose
129
None
130
Mannitol
131
Trehalose
132
Mannitol
133
Sucrose
134
Trehalose dihydrate and sorbitol
3240 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
According to the results, a critical freezing rate, below which the
dried NCs cannot be re-dispersed, was identified based on the plot
of the PS of reconstituted NCs versus freezing rate. When the
freezing of dispersion was carried out in a slower rate, the trap of
adsorbed polymers or fusion of API crystals, probably led to robust
aggregations. On the other hand, when freezing rate was close to its
critical rate, bimodal PS distribution in re-dispersed formulation
emerged showing singular particles along with the aggregates. It
was confirmed that the concentration of NPs has an impact on the
critical freezing rate because when the concentration of NPs
reduced from 11.5 to 6.1 wt %, the critical freezing rate decreased
several times. When the dispersion concentration was 1.8 wt %, the
critical freezing rate was simply so low that was impossible to be
measured by the present equipments. Usually, reducing the con-
centration directly results in a rise in inter-particle distance and a
decrease in density of NPs which reduces the collision frequency of
NCs and decelerate the aggregation kinetics.69 Moreover, the sol-
vent/antisolvent ratio through freeze-drying stages (both freezing
and drying) is very important. It has been discovered that by uti-
lizing a solution with lower water/tertiobutyl alcohol (TBA) ratio in
the freezing step, quicker dissolution was resulted showing that the
crystalline dispersions included smaller crystals. When crystalli-
zation happened through drying, employing solutions with higher
water/TBA ratio led to the composition of finer solvent crystals.
Accordingly, two variables of freezing rate and water/TBA ratio
should be considered to modify the crystal size. When crystalliza-
tion happens in freezing phase, a higher nucleation rate might
result from faster freezing or smaller water content of the solution.
As a result, a lot of nuclei are shaped leading to smaller crystals.70 At
the same time, crystallization in drying phase might additionally
happen when the temperature increases higher than glass transi-
tion temperature of the maximally freeze-concentrated fraction
(Tg') and below the eutectic temperature (Te). In such a case, what
is obtained is the rubbery state of the freeze-concentrated fraction
and crystallization will happen. The crystals enlargement is limited
to the interstitial areas extent between the solvent crystals. The
researchers showed that the interstitial areas' dimensions between
the solvent crystals including the freeze-concentrated fraction may
be altered by modifying the rate of freezing or the water/TBA ratio.
When the rate of freezing was slow or there existed a high water/
TBA ratio, large solvent crystals formed having large inter-crystal
spaces. However, smaller solvent crystals are going to be formed
when the solutions were rapidly frozen (or when there exists a
minimum amount of water in the solution), leading to tiny inter-
stitial areas. As the crystallization happens within the freeze-
concentrated fraction, the ultimate dimensions of drug crystals
can be limited by the interstitial areas which exist between the
solvent crystals.70 The researchers defined a re-dispersibility index
(RDI) as D0/D where D0 and D represented the volume-weighed
mean PS of the freshly prepared of ursodeoxycholic acid NC
(UDCA-NC) and final PS after freeze drying, respectively. The RDI of
UDCA-NC depended on both the intensity of freezing and the type
of stabilizers. The various freezing temperatures might lead to the
irrevocable aggregation of UDCA-NC throughout freezing step. As
an example, RDI 20 C, RDI 80 C and RDI 196 C of UDCA-NC stabi-
lized by 10% w/v of PVP 30 K were 2.98, 9.01 and 14.16, respectively.
However, those of UDCA-NC which have been stabilized by 50% w/v
of PVP 30 K was 1.16, 5.32 and 9.65, accordingly. The mentioned
outcomes signify that the dispersibility of frozen UDCA-NC at the
standard freezing degree (meaning low freezing rate) was higher
compared to the moderate and high temperatures (high freezing
rate).71 In another study it was declared that the re-dispersibility of
harmine solid NCs (HAR-NCs) at the rigorous conditions (the
highest freezing rate) was higher compared to the conservative and
moderate states. The probable explanation by authors was that the
external particles are kept out by molecules of water when HAR-
NCs having adequate time and also making them come close to
each other and this finally leads to aggregation at low freezing rate
(conservative and moderate state), and at the same time, the sta-
bilizers' activity proves to be ineffective due to the phase detach-
ment.72 The best freezing rate may give suitable conditions to stop
NCs from aggregating or perhaps prevents the fusion of NCs
through the freezing step.69 In the case of dosage forms that are
dissolved in mouth, low freezing rate is highly acceptable because
structures with giant pores are formed, which can be simply dis-
integrated in the presence of hydration. Although disintegration is
fostered by giant pores, when we deal with the systems including
suspended NCs, a dense cryo-concentrated stage through freezing
may prompt aggregation.73 An annealing step has been conjointly
noted for the mentioned advantages such as ice crystals' size
dispersal speeding up initial drying and reducing heterogeneity
among samples.38 Furthermore, mannitol as the bulking agent was
discovered to give a mix of amorphous and crystalline forms.74
Particularly, when mannitol to drug ratios are low, crystallization
might be optimized if an annealing stage is merged into the
freezing stage.75 The kind of freezing influenced the disintegration
annealing and diminished the disintegration period whilst it was
deferred by progressive freezing. Strangely enough, the product's
hardness or fracturability were merely influenced by freezing pa-
rameters (it might be noted that a product's fragility is indirectly
shown by fracturability and is measured according to the primary
fracture's load value. More specifically, it indicates a product's
endurance toward fracture).73 Dissolution is postponed by sharp
freezing in contrast to the progressive freezing and therefore, it
appeared to receive a less considerable intensifying influence by
the annealing. As a result of poor mechanical force, the act of
dissolution occurred quicker by slow freezing and therefore, ice
crystals increase led to a raise in the porosity within the freezing
phase.76
Stabilizer Effect
The size variations of NCs caused by lyophilization with PVP
stabilizers showed separate affirmative impacts for PVP as a sta-
bilizer. It meant that at low concentrations, PVP caused a growth in
the size of NCs. The PVP to drug ratio being significantly low, in-
creases the possibility of the particle's surface not being fully
covered by adsorption.77 In meloxicam NCs, the areas of the
meloxicam particles which were not covered, might display ag-
gregation capability and caused a moderate raise in the size. An
interactional impact between PVP as stabilizer and the concentra-
tion of stabilizer demonstrated as a decrease in size which was
resulted from high PVP content, confirms the previous hypothesis.
Accordingly, poloxamer in the varying concentrations produced an
affirmative small impact on the size of crystals. It induced a
decrease in PS right down to 10.27% of the primary size. In contrast,
a remarkable reduction in size was obtained from lyophilizing of
nanosuspensions with PEGs as stabilizer.76 As poloxamer led to
lowering of PS in addition to protect meloxicam crystallinity, the
maximum reduction was observed in the formulations containing
1% poloxamer and 5% mannitol prior to lyophilization.73 The type
and quantities of stabilizers in the same freezing method had
dramatically significant diverse protection impacts on UDCA-NCs.
Higher the concentration of stabilizers were, better re-
dispersibility was obtained. It suggested that the ability of being
re-dispersed in UDCA-NCs prepared by 50% w/v concentration of
surfactants or polymer stabilizers turned out to be far superior to
the others with 25% and 10% w/v concentrations. Also, the results
showed that the surfactants (TPGS, Cremophor RH40 and Tween
80) proved to be more efficient in comparison with polymeric
stabilizers (HPMC, NaCMC).71 The same results were obtained
 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
about harmine solid NCs (HAR-NCs). It was reported that re-
dispersibility of HAR-NCs having high (50% w/v) concentration of
surfactants or polymer stabilizers were largely superior to the
others with moderate and low concentrations. Furthermore, the
quantity and type of stabilizers significantly influenced the re-
dispersibility of HAR-NCs after freeze-drying.72 It may be reason-
able that HAR-NCs might approach to each other through crystal
bridges when drying and they merge to form big agglomerates.78
The polymeric stabilizers appeared to be adequately efficient to
guard the HAR-NCs during varying stresses throughout drying
which might be due to their homogeneously adsorption onto the
top layer of HAR-NCs and forming a steric barrier layer.72
Cryoprotectant Type and Concentration
The type and concentration of cryoprotectants compete for a
vital role in maintaining smart re-dispersibility options of NCs. To
set the criterion of the freeze-drying cycle, initial evaluations of
cryoprotectant and Tg values were carried out for all cryoprotectant
solutions and a cryoprotectant blended with nanosuspensions
too.79 Determining Tg for cryoprotectants separately and also in
form of a mixture with nanosuspension indicated that effective
cryoprotection was obtained by determining the smallest change in
Tg values. In that sense, six types of sugars or polyols (glucose,
maltose, sucrose, lactose, mannitol, and sorbitol) (3%, w/v) in the
role of lyophilization supports have been studied in preparation of
azilsartan NCs. Sucrose was the foremost effective lyophilization
support and induced highest stability on lyophilization method.80 It
was stated that by decreasing the mannitol's concentration to 5%
and 2% w/v, the system became unstable. The results showed that at
least 10% of mannitol and other protectants was required for pos-
sessing a proper PS ratio.81 Moreover, it has been revealed that RDI
of UDCA-NC when 10% w/v of cryoprotectants (like lactose and
mannitol) was added to formulation was more than 2 whereas
adding 400% w/v of cryoprotectants (for instance sucrose, sorbitol)
showed RDI of less than 2.71 Moreover, it was observed that cryo-
protectants sorbitol and sucrose had higher efficiency on RDI of
HAR-NCs compared to other protectants. Also, the more the cryo-
protectants' concentration increased, the higher protection under
all process conditions was observed. These observations were
supposed to be related to osmotic pressure effects. On the other
words, these results could be explained by comparatively high
concentrations of adsorbed polymers or sugars within the area
between both surfaces when they get closer to each other.72 A
previous study showed the importance of the treatments before
lyophilization on cellulose NCs (CNC) morphology. The researchers
demonstrated that CNCs freeze-dried from 1% suspension had
smaller flakes compared with CNCs freeze-dried from 10.7% sus-
pension. However, sonication and flash freezing of the CNC sus-
pensions resulted in fibrillary structures within the freeze-dried
CNC morphology. This caused to a much higher degree of crystal-
linity. Consequently, the agglomerate sizes were decreased by
dilution and/or increasing sonication time.82
Relationship Between Polymer Stabilizer and Cryoprotectants
It has been considered that the ability of NCs for re-dispersion
may be related to a synergistic effect between polymer stabilizer
and cryoprotectants. It has been found out that during the freezing,
the cryoprotectants solution becomes freeze-concentrated and
therefore produces a stable glass which is highly viscous. This
glassy state has minor mobility preventing NCs from fusion/ag-
gregation and protects them against ice crystals. Lastly, the
hydrogen bonds of sugar reduce the interplays between the water
and UDCA-NCs and replace those of water in freeze-dried prod-
uct.71,83 Moreover, the probable interplays between the head
groups of sugars and polymeric stabilizers reduce the trap of
3241
polymer chains within the dry stage and prevent the polymer
stabilizer's steric barrier effect.83 It was additionally showed that at
10% w/v concentration of TPGS, Tween 80 and HPMC as stabilizer,
RDI of HAR-NCs in the presence of 400% w/v concentration of su-
crose, trehalose and sorbitol was closer to one in comparison with
100 and 200% of them. Furthermore, 100% w/v concentration of
sucrose, glucose or sorbitol had a better function on HPMC stabi-
lized HAR-NCs compared with alternative cryoprotectants.72
Moreover, NCs that have been made stable with Pluronic® F-68
or Pluronic® F-127 indicated more growth in PS in the presence of
cryoprotectants. As a similar result, other researchers reported that
the NCs containing solely SLS possessed smallest PS ratio.81
Solvent Effect
Solvents that do not freeze and/or sublime fully using industrial
freeze dryers have been proved to be harder to be employed and
sometimes led to freeze-dried intermediates which were not
satisfactory. By choosing solvent DMSO, the freeze-drying of gli-
benclamide NCs was unacceptable because the freeze-dried in-
termediates happened to be greasy, yellow, and sticky and the
solvent features of DMSO could describe that. DMSO is a solvent
with high freezing point that is suitable for a decent freeze-drying
method having really a low vapor pressure.84 For evaluating the
solvent effects, various solvent mixtures were put into trial con-
sisting of DMSO (major solvent, reasonable solubility, low quality
freeze-drying outcomes) and TBA (secondary solvent, low solubil-
ity, reasonable freeze-drying outcomes) in diverse solvent ratios to
enhance freeze-drying functionality. Characteristics such as high
freezing point, high vapor pressure and low toxicity have made TBA
a suitable solvent for freeze-drying. Using TBA in the role of an
organic co-solvent resulted in obtaining a satisfactory freeze-
drying performance. A permeable and fluffy product was ob-
tained when TBA was used as the supplement solvent.85 The su-
perior freeze-drying outcomes (fluffy cakes) were achieved with
the DMSO: TBA ratios of 25:75 and 10:90. The amount of the
organic solvents throughout the freeze-drying method has proved
to be vital in changing the API's crystalline form to an amorphous
one. With DMSO:TBA 25:75, the drug crystal behavior changed
from crystalline to amorphous, however too much TBA (for
example the DMSO: TBA 10:90 sample) caused to an increase in
crystalline behavior.84
Nanoemulsions
NEs known as oil-in-water (O/W) or water-in-oil (W/O) are
dispersions of two immiscible fluids being stable by use of an
acceptable surfactant.86 The mean droplet diameter appears to be
less than 500 nm.87 The small droplet size especially those less than
100 nm provides them with a transparent or blurred look that
varies from milky white color related to large emulsions (which
micron-sized droplets take part in numerous light dispersions).88
NEs despite having a similar size differ from microemulsions
enormously in structural characteristics and the thermodynamic
stability in the long time.89 The mentioned long-lasting physical
stability may directly be the result of small droplet size that pro-
hibits typical destabilization occurrences such as creaming, sedi-
mentation and coalescence. Usually, brownian motion appears to
be adequately powerful to balance gravity or viscosity-evoked dy-
namic instability.90 NEs are at risk of one major instability phe-
nomenon called Ostwald ripening. The mentioned phenomenon is
accountable for the increase in NEs' droplet size, by means of
inducing instability and it can be typically related to the system's
aging. The event happens similarly in typical emulsions. Ostwald
ripening could also be controlled via the modification of the
droplets size and distribution. Using NEs is usually favored to
3242 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
microemulsions though they show more instability because the
concentration of their surfactants are remarkably lower.91 Recently,
a study revealed that the droplet size of self-nanoemulsifying drug
delivery systems (SNDDSs) increased when the freeze-drying
method was applied. Nevertheless, this growth in the size was
tolerable as the droplet size remained below 200 nm but other
physicochemical characteristics like polydispersity index (PDI),
conduction and zeta potential, remained the same.92 The freeze
drying of these formulations is influenced by the type and con-
centration of surfactants. When the unsaturation temperature is
low, the surfactant's acyl chains tend to be longer and when the
phase transition degree of the lipid is higher, additional drug loss is
resulted.93 Although the emulsions, NEs and microemulsions might
be precisely formed from the same ingredients, the microemulsions
need a higher surfactant to oil ratio.94 Choosing the type and the
concentration of the cryoprotectant for NEs is vital. When the
cryoprotectant concentration is adequate, it can interrelate with
the surface of the droplets and protect them through freeze-drying.
The most of cryoprotectants used in lyophilization of the emulsified
systems are disaccharides and saccharides. Amongst them, treha-
lose has appeared to have the highest efficiency. This superiority is
the result of its low hygroscopicity, ability to create inter-molecular
hydrogen bonds, small chemical reactivity as well as high Tg.95 The
perfect cryoprotectant concentration for emulsified systems fluc-
tuates between 5% and 20% w/v.96 The study on freeze-dried
bufadienolides-loaded NEs (BU-NE) indicated that the protecting
efficiency of maltose, trehalose and sucrose alone and in combi-
nation was remarkable whereas glucose wasn't effective. These
excipients were the foremost effective at the identical concentra-
tion of 20% w/v. Ironically, there was a rise in particle aggregation in
the presence of cryoprotectants with higher concentrations. In-
stances including trehalose/sucrose and maltose/sucrose combi-
nations of varied compositions did not show to further improve
cryoprotective effect. The influence of various cryoprotectants
(glucose, fructose, mannitol, sucrose, lactose, trehalose and
maltose) on bufadienolides-loaded submicron-emulsions (BU-SE)
has been investigated. The existence of an exact quantity of
amorphous disaccharides in lyophilization proved to be an essen-
tial issue for rehydrated BU-SE. The rehydrated BU-SE including
mannitol, glucose, fructose, and lactose did not reveal a Tindall
effect. The lyophilization results suggested that the best cryopro-
tectants for BU-NE and BU-SE have been maltose and trehalose at
an identical concentration of 20% w/v.97 Adding cryoprotectant to
the NEs was accomplished after the completion of the process. The
results demonstrated differences in NE features such as droplet size
and drug loading capacity, due to adding cryoprotectant at various
stages. Adding cryoprotectants in the step of preparing NEs led to
an increase in droplet size and polydispersity. However, when the
researchers tried to add the cryoprotectants in the last formulation,
it was troublesome to dissolve a number of the sugars like mannitol
and lactose. Also, a rise in the droplet size and drug leaking would
be resulted in the case of using ultrasound.97
Generally, the freezing states ought to be cautiously identified.
In slow freezing, as the water will diffuse in a slow rate, the osmotic
pressure is decreased stopping the outpouring of the droplets.98
Nevertheless, the application of average freezing rates may well
Table 3
The Examples of Drug Nanoemulsions Manufacturing Methods and Main Excipients Used for Freeze-Drying.
Compound Method
Febuxostat Self Nanoemulsifying Drug Delivery Systems
Ginkgo biloba Emulsion solvent evaporation
Bufadienolides Ultrasonic-high-pressure homogenization
Adefovir dipivoxil Self Nanoemulsifying Drug Delivery Systems
be beneficial, as it will not let the system freeze extraordinarily and
would keep it from destruction. Furthermore, it maintains the rate
of freezing adequately high to ease secondary drying.96 The product
temperature ought to be near to the Tg, since the enhancement of
the temperature leads to the quicker procedure as some re-
searchers reported that a rise of one centigrade degree in the
temperature of the product decreased the first drying time by
thirteen fold.21 NEs probably have the shortest primary-drying time
as a result of containing the biggest proportion of water which
leads to a quicker mass transfer. The secondary-drying starts by a
slow-paced rise in the shelf temperature (0.1 or 0.15 C/min) since a
quick temperature change may result in the destruction of the
amorphous products due to having high unused humidity and
possessing a low Tg.21 Further, the instances with higher amount of
particles dissolved in them, maintain smaller space within the dry
state. Consequently, higher temperature and an extended period is
required to get rid of the water.98 Table 3 summarizes the freeze-
dried NEs found in literature including the manufacturing
method and main excipients used to optimize freeze-drying.
Nanocapsules and Nanospheres
NCPs consist of colloidal dispersions exhibiting a core-shell
structure which the core functions as a fluid reservoir for medi-
cines. The structure is usually composed of polymers, desirably
decomposable, thick and stiff in the nanometric scale. The benefits
of this kind of formation can be as follow; first, NCPs have high
medicine encapsulating efficiencies because of the maximized
solvability of drug within the NCP core and small polymer amount.
Next, as the medicine is preserved in the NCP core and remains safe
from degradation. Furthermore, tissue irritation due to the burst
effect at the administration site is low.99 Numerous strategies have
been sometimes employed for creating this kind of core-shell
construction on a nanometric scale including in-situ polymeriza-
tion at the NE droplet interface, NCP generation by use of pre-
formed polymer and also generating of lipid NCPs. Lipophilic and
hydrophilic parts of the NCPs are distinguishable provided that the
NCPs are dispersed in water.100
On the other hand, a polymeric NSP could be described as a
matrix kind, firm colloidal particle which in drugs are solved,
trapped, or enclosed with the chemicals bound or adsorbed onto
the polymer matrix. The mentioned particles are generally bigger
compared to micelles which have diameters ranging from one
hundred to two hundred nanometer and usually show significantly
higher polydispersity.101
Freeze-drying is thought-about as a decent methodology which
preserves the unification of colloidal dispersions. Thus, the freeze-
drying of NCPs and NSPs which possess slim and frangible structure
barely resistant to the mechanical force of the process has special
considerations. Therefore, it is important to examine the features
which may have an effect on the NCPs and NSPs stability
throughout the various stages of lyophilization. Table 4 summarizes
the freeze-dried NCPs and NSPs found in literature including the
manufacturing method and main excipients used to optimize
freeze-drying.
Aid Excipients for Freeze-Drying Literature Reference
135
Trehalose
136
Hydroxy beta cyclo dexterin
97
Maltose and trehalose
92
D-mannitol
 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
Table 4
The Examples of Drug Nanocapsules and Nanospheres Manufacturing Methods and Main Excipients Used for Freeze-Drying.
Compound Method
Vitamin E Nanoprecipitation
Chitosan containing Docetaxel Solvent displacement
Capsicum Emulsionediffusion
Itraconazole Emulsion solvent evaporation
Mitoxantrone Nanoprecipitation/solvent diffusion
Poly(ester-anhydride) Water-in-oil-in-water double emulsion
Methacrylic acid Solution/precipitation polymerization
Ketorolac tromethamine Microreactor technique
Freeze-Drying Cycle Effect
About NCPs, diverse dispersions were discovered at completely
distinct places within the freeze-dried bulk sample, and this non-
homogenity has been obsessed with the cooling program utilized
within the process. As an instance, when gelatin NCPs mixture's
temperature was quickly reduced and it was frozen, bottom layers'
dispersibility was lower compared to the layers at the top, and this
tendency appeared more obvious at the external portions. In
another example, when freezing of the sample suspensions took
place at a continual slow cooling rate, dispersions had a very small
nonhomogenity within the dried bulk sample. In addition, in
comparison with fast cooling it had a better dispersibility. It was
discovered that there is a mutual relationship between gelatin
NCPs' dispersibility and the freezing front speed, and therefore with
the viscosity at or around the freezing front. It has been recom-
mended that the forming of gel is beneficial to prevent undesirable
denaturing and to provide better dispersion within the dried ma-
trix. Nevertheless, slow dehydrating might result in extra stress due
to the development of enormous ice crystals resulting in low dis-
persibility within the ultimate dried product.102 The lower freezing
temperatures were additionally effective for keeping the initial size
and morphology of NPs. The mean PS of freeze-dried fish oil NCPs
was considerably raised by reducing the freezing temperature.
Therefore, to optimize the lyophilization procedure, higher freezing
temperature was the most favorable state to keep up the fish oil
quantity within the particles and delay oxidation.103 It has been
demonstrated that the sublimation rate increased and the hetero-
geneity of the sample decreased by annealing.104 Annealing had an
effect on initial drying by speeding up the sublimation rate with no
change in the size of NCPs. The sublimation rate of NCPs utilizing
sucrose and PVP as protectants showed the sublimation rate of
87.9 mg/h for sucrose and 70 mg/h for PVP. However, annealing
produced the best sublimation rates. Using sucrose, the sublima-
tion rate increased from 86.7 to 124.8 mg/h, while about PVP, the
sublimation rate rose from 77 to 83.9 mg/h when the annealing
temperature went up from 20 to 10  C.50 These findings indi-
cated that accelerating the sublimation rate relied on the annealing
temperature.
The impact of annealing on secondary drying depends on the
type of cryoprotectant employed. Studies showed that annealing of
sucrose solution slowed down the secondary drying kinetic while
there was not any impacts using PVP. Sucrose seems to be slightly
less hygroscopic compared to PVP. This sort of dissimilarity in hy-
groscopicity could be responsible to the difference between the
secondary drying kinetics of the compounds.105 Searle's, Carpenter
and Randolph indicated that following an annealing process, the
drying rate might rise up to 3.5 fold and only 30 min of annealing
could improve the rate significantly.104 It has been found that by
annealing of ovalbumin loaded carboxymethyl chitosan NCPs
at 15  C, the NCPs sizes did not considerably change and therefore,
the ratio of NCPs sizes pre and post annealing was close to one.
Although, when the procedure was carried out at 25  C, NP size
ratio was raised, the ratio of the NCPs PDI pre and post annealing
3243
Aid Excipients for Freeze-Drying Literature Reference
137
Sucrose
138
Trehalose
139
Pluronic F68
108
Sucrose, Glucose, Lactose, Trehalose, Mannitol
140
Lactose
141
Trehalose
142
Pluronic F68
143
Mannitol
was 1.15 which is acceptable. Also, the procedure conducted
at 15  C, 10  C, and 5  C showed that a rise in the ratio of NCPs
PDI pre and post annealing was 1.68, 1.92, and 2.31, accordingly.106
Formulation Effect
The cryoprotectant nature and concentration have vital roles in
the lyophilization process. Disaccharides which are stable chemical
additives proved to be beneficial excipients able to vitrify simply
throughout the freezing stage.36 The characteristic disaccharides to
make a glassy matrix within which the NPs are fixed, prevent the
particles from aggregating.107 Disaccharides such as sucrose and
trehalose appeared to be consistent with polybutyl cyanoacrylate
(PBCA) and thus functioned as perfect cryoprotectants for PBCA-
NSP loading itraconazole. Glucose and lactose failed to stop itra-
conazole leakage when reconstitution.108 Same as mannitol, lactose
tends to crystallize more even within the freeze-drying cake in
comparison with alternative disaccharides.36 This will result in
phase division and therefore the NPs complicated structure can be
damaged. As the researchers reported, the freeze-dried cake con-
taining 10% glucose fell down and it was rather hard to suspend the
lyophilized cake again. It seems that in this case, the collapse
temperature (CT) went over and caused to structural collapse
within the dried matrix and itraconazole leakage when reconsti-
tution. Moreover, it may well be revealed that the cryopreservation
quality of disaccharides like sucrose and trehalose is related to their
concentration inside the nano-formulation. A cryoprotectant con-
centration lower than 10% was not enough to preserve the original
itraconazole loading and construct a preventing vitrification layer
around the PBCA-NSPs. For the long term, the researchers have
used trehalose as choice cryoprotectant because it is highly stable
in conditions with high temperature and humidity in comparison
to sucrose. This feature is often due to the higher Tg of dried
trehalose matrix when adsorbing vapor.108 Studies revealed that
the dominant destabilization cause in freeze-drying of NSPs was
ineffective surface modifications and significant drug desorption.
Therefore, the hydrophilic surfactants adsorbed at NSPs surface had
a great impact on cryopreservation. Replacement of the normally
employed non-ionic surfactant pluronic F68 by the anionic sur-
factant sodium deoxycholate led to a very successful stabilization of
itraconazole-loaded NSPs without desorbing the drug, albeit when
10% sucrose was present and not glucose. Pluronic F68 might
crystallize through freezing leading to surface alternations and
drug desorption whereas this might not happen with sodium
deoxycholate.109 It may be observed that if the stabilizer's con-
centration is raised, a decrease in the size and polydispersity of
NSPs is resulted. The researchers showed that when the PVA con-
centration as stabilizer was raised from 0.62 to 5% w/v, the ratios of
NCPs size after and before lyophilization (SF/SI) decreased from 4 to
1 indicating that the initial size was remained unchanged. The PVA
concentration of 2.5% w/v was the lowest of which was needed for
stabilizing the NCPs when freezing. Similarly, about poly (epsilon-
caprolactone) (PCL) NCPs, 2.5% w/v of sucrose was the lowest
concentration required to enhance NCPs stability. Furthermore, the
3244 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
increase of PCL concentration induced a rise of size though the
calculated envelope thickness was in the accepted range. The trial
of various concentrations of PCL demonstrated that the concen-
tration of PCL did not enhance the NCPs stability when freezing
probably due to the increase of NCPs size. The freeze-thawing
stability of PCL NCPs was obtained from two oils (miglyol and sil-
icon oil). Consequently, preserving the enclosed oil in the liquid
condition did not increase the stability of NCPs in the freezing step.
Thus, it may be assumed that the stabilizer proportion utilized in
the formulation and also cryoprotectant are the foremost vital el-
ements that affect the stability in the freezing step.50 These fragile
particles may be destabilized in the freezing or the dehydration
steps owing to the crystallization of for instance mannitol. Under
storage time, NCPs freeze-dried in the presence of sucrose and
glucose may well be aggregated if exposed to high temperatures
and moderate humidities. The mentioned state might begin the
crystallization of amorphous lyoprotectants inducing aggregation
of NCPs. Therefore, freeze-dried NCPs ought to be kept at a tem-
perature lower than the glass transition temperature of the
formulation to keep up the glassy condition of lyoprotectant and to
stop aggregation of NCPs. These findings were related to the NCPs
which have been freeze-dried with PVP and preserved their sta-
bility for only six months of being stored in stress conditions.29
It is worthy to mention that the characteristics of the bio-
polymers and crosslinking method have made the direct freeze-
drying of NCPs possible without the need to use common poly-
ols cryoprotectants. The biopolymers having global structure will
stick onto the surface of NCPs with high affinity via different
crosslinking methods forming namely a “rigid wall” preserving the
lipid core. As it was indicated in the preparation of NE-templated
shell-crosslinked NCPs of fenofibrate, when the aqueous
biopolymer was freeze-dried, these biopolymers were glassified
forming a lyocake having a suitable quality without showing the
indications of collapse. These observations propose that the free
biopolymers themselves will work in the role of cryoprotectants
preserving the product against freezing and drying stresses.110 The
restoration of the primary characteristics of chitosan NCPs on
lyophilization and reconstruction was different based on the
concentration of NCPs (0.25, 0.5 and 1% w/v) and cryoprotectant (5
and 10% w/v). The most effective outcomes were achieved for a
NCP concentration of 0.25% w/v and trehalose concentration of
10% w/v. The freeze drying of chitosanepoloxamer NCPs in pres-
ence of 10% w/v of trehalose indicated a 50e100 nm rise in the
size of NCPs which can be attributed to higher crystallinity of
glassy matrix structure in the presence of poloxamer. This
augmented crystallinity might be the reason of the lower
Table 5
Key Considerations in Freeze Drying of Different Classes of Nanoparticles Including Formulation Parameters and Optimum Freeze-Drying Conditions.
Nanoparticles Classifications Most Influential Formulation Parameters
Nanosuspensions Stabilizer structure and conc.
Cryo/lyoprotectant type and conc.
NPs conc.
Nanocrystals Cryo/lyoprotectant Tg and conc.
Stabilizer-cryoprotectant interaction
Solvent characteristics
Solvent to anti-solvent ratio
NCs conc.
Nanoemulsions Stabilizer structure and conc.
Cryo/lyoprotectant conc.
Nanocapsules/Nanospheres Stabilizer structure and conc.
Shell crosslinking degree
Cryo/lyoprotectant type and Conc.
NCPs conc
stabilizing impact of trehalose. Accordingly, the lyophilization of
docetaxel-loaded chitosan NCPs without poloxamer revealed that
the freeze-drying of drug-loaded NCPs might take place with the
identical quantities of trehalose and the resulting freeze-dried
product might be properly reconstructed with no loss of for-
mulation's characteristics.111 Another team of researchers studied
the effect of different concentrations of Hyaluronan NCPs (0.5 and
1% w/v) and trehalose (5% and 10% w/v) using benzalkonium
chloride as the surfactant. The results showed that at rather high
concentrations of NCPs (1% w/v), it was likely to attain an
appropriate re-suspension of the freeze-dried product without
significant change in the size of the NCPs.112
Cocrystals/Nanococrystals
Cocrystals are in fact “homogenous” (single phase) crystalline
forms created from two or more compounds possessing a distinct
stoichiometric quantitative ratio wherever the positioning within
the crystal lattice does not rely on ionic bonds.113 Cocrystal
structure relies on noncovalent interplay of the API and also the
components known as conformer. The involving interactions are
intermolecular ones like hydrogen bonding, van der Waals con-
tact forces, p stacking, static interaction and halogen bonding
among stoichiometric quantities of assorted molecules.114 Coc-
rystallization strategies are classified based on the employment
(or not) of solvent, so the cocrystallization methods are catego-
rized as solvent-based or solvent-free. Solvent-based strategies
are the most routine and accepted ones particularly for laboratory
small-sized utilization. The mentioned strategies can sometimes
be unsafe due to using considerable quantities of hazardous
solvents.115
Freeze drying may be a technique usually resulting to amor-
phous substances though it can be sometimes appropriate for
producing cocrystals too. For producing cocrystals, certain molar
quantities of the compounds are dissolved in a suitable solvent. The
solvent choice is performed according to instrument chemical
compatibility and solvent capability for dissolving the API and
coformer efficiently. The resulting solution is subjected to a quick
freezing and also a high vacuum which results in subliming the
solvent. A low-density powder is created, typically in amorphous
form. If the glass transition temperature of the product is lower
than ambient temperature, amorphous forms are prone to form.
However, cocrystallization with this methodology does not have
any kinetic obstacles to produce crystalline forms.116 For instance,
with dissolving theophylline and various coformers in water or
tertiobutanol as solvent, the cocrystals of many forms were
Optimum Freeze-Drying Conditions
Slow/fast freezing rate
Short primary drying Period
Fast freezing rate (most often)
Short primary drying period
Moderate freezing rate
Higher temperature in freezing/drying periods
Extended freezing/drying periods
Short primary drying period
High/low freezing temperature
Application of annealing
Short primary drying period
 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
obtained including completely unique solid solution of theo-
phylline:caffeine and a possible new cocrystal kind of theophylli-
ne:oxalic acid. X-ray powder diffraction (XRPD) and DSC analyses
showed the existence of a pentahydrate theophylline:oxalic acid
2:1 cocrystal for the freeze-dried instances. About the theophylli-
ne:caffeine, the analyses demonstrated the same paradigm; how-
ever, lack of an amorphous form that would be ascribed to the
single component phase of theophylline indicated a solid solution
of the two constituents. Additional examinations demonstrated the
forming of single NeH bond between the two molecules.117 How-
ever, the mentioned methods additionally had a number of disad-
vantages. They needed adequate API and coformer solubility in
appropriate solvents, particularly water and t-butanol. In addition,
when solidifying is fast, amorphous solids or crystals with less
stability might be produced.
Freeze-drying is appropriate for the sterile producing of solid
formulations for injection.118 Main advantages of cocrystal
manufacturing by freeze-drying include being single step and
continuous process, easy scale up, and prevention of single-
compound crystals formation.116 Freeze-drying might be helpful
in many phases of cocrystal formation from primary cocrystal
screening and examination of cocrystal polymorphism to prepa-
ration of cocrystals in industrial scale.117
Although the co-crystal and nanocrystal technologies have been
widely used in the development of pharmaceuticals, but their
synergistic effect has not been well investigated. Nanococrystals,
which combine benefits of both technologies can be used to solve
the problem of water-insoluble drugs. Therefore, similar to nano-
crystals, lyophilization can be used to produce solid forms of
nanocorystals. Athough there aren't many studies on these systems,
it seems that the same considerations as nanocrystals could be
imagined for nanococrystals too.
Table 5 summarizes the most influential parameters related to
NPs formulation and the most optimum freeze drying conditions in
which the best results would be achieved.
Conclusion
The lyophilization procedure is of great importance for
improving the long-term stability of NPs. Therefore, the therapeutic
goals and specifications of the drug must be considered for
lyophilization. To achieve a good product, there exist numerous
factors in NPs formulation and also freeze-drying process which
need to be optimized. The most influential factors include the type
and concentration of cryoprotectant and stabilizer, the relationship
between protectant and stabilizer, the concentration of NPs,
freezing, annealing, and drying rate. Without these considerations,
the resulting freeze-dried product may not be able to reproduce the
primary NPs dispersions regarding their PS and PDI. However, the
freezing of NPs encounters the challenges that need further
research to overcome. One of these is the scale-up considerations
and physical stability of lyophilized NPs to reach to the pharma-
ceutical market. In this regard, the theoretical mathematical
models can be employed to design, control, optimize and scale-up
different phases of freeze-drying processes. Also, lyophilization can
be used in the future to process the new developing types of NPs
like nanotubes, nanofibers, and nanoaggregates in industrial scale
provided that these delivery systems could meet the safety re-
quirements for using in humans.
Ethics Approval and Consesent for Publication
Not applicable.
3245
Conflicts of Interest
The authors declare no conflict of interest, financial or
otherwise.
Acknowledgements
The authors would like to thank pharmaceutics laboratories of
Shiraz School of Pharmacy for providing detailed information about
freeze-drying instrument.
References
1. Shi J, Votruba AR, Farokhzad OC, Langer R. Nanotechnology in drug delivery
and tissue engineering: from discovery to applications. Nano Lett. 2010;10(9):
3223-3230.
2. Sung JC, Pulliam BL, Edwards DA. Nanoparticles for drug delivery to the lungs.
Trends Biotechnol. 2007;25(12):563-570.
3. Gao L, Zhang D, Chen M. Drug nanocrystals for the formulation of poorly
soluble drugs and its application as a potential drug delivery system.
J Nanoparticle Res. 2008;10(5):845-862.
4. Desai MP, Labhasetwar V, Walter E, Levy RJ, Amidon GL. The mechanism of
uptake of biodegradable microparticles in Caco-2 cells is size dependent.
Pharm Res. 1997;14(11):1568-1573.
5. Fonte P, Araújo F, Reis S, Sarmento B. Oral insulin delivery: how far are we?
J Diabetes Sci Technol. 2013;7(2):520-531.
6. Courrier H, Butz N, Vandamme TF. Pulmonary drug delivery systems: recent
developments and prospects. Crit Rev Ther Drug Carrier Syst. 2002;19(4-5).
7. Lemoine D, Francois C, Kedzierewicz F, Preat V, Hoffman M, Maincent P.
Stability study of nanoparticles of poly (e-caprolactone), poly (d, l-lactide) and
poly (d, l-lactide-co-glycolide). Biomaterials. 1996;17(22):2191-2197.
8. Wu L, Zhang J, Watanabe W. Physical and chemical stability of drug nano-
particles. Adv Drug Deliv Rev. 2011;63(6):456-469.
9. Chacon M, Molpeceres J, Berges L, Guzman M, Aberturas M. Stability and
freeze-drying of cyclosporine loaded poly (D, L lactideeglycolide) carriers. Eur
J Pharm Sci. 1999;8(2):99-107.
10. Auvillain M, Cave
G, Fessi H, Devissaguet JP. Lyophilisation de vecteurs col-
loïdaux submicroniques. STP Pharma. 1989;5(11):738-744.
11. Katas H, Hussain Z, Rahman SA. Storage stabilisation of albumin-loaded chi-
tosan nanoparticles by lyoprotectants. Trop J Pharm Res. 2013;12(2):135-142.
12. Boge L, et al, V€ astberg A, Umerska A. Freeze-dried and re-hydrated liquid
crystalline nanoparticles stabilized with disaccharides for drug-delivery of the
plectasin derivative AP114 antimicrobial peptide. J Colloid Interface Sci.
2018;522:126-135.
13. Chung N-O, Lee MK, Lee J. Mechanism of freeze-drying drug nanosuspensions.
Int J Pharm. 2012;437(1-2):42-50.
14. Bozdag S, Dillen K, Vandervoort J, Ludwig A. The effect of freeze-drying with
different cryoprotectants and gamma-irradiation sterilization on the charac-
teristics of ciprofloxacin HCl-loaded poly (D, L-lactide-glycolide) nano-
particles. J Pharm Pharmacol. 2005;57(6):699-707.
15. Lu X, Pikal MJ. Freeze-drying of mannitoletrehaloseesodium chloride-based
formulations: the impact of annealing on dry layer resistance to mass transfer
and cake structure. Pharm Dev Technol. 2004;9(1):85-95.
16. Wong JJL, Yu H, Hadinoto K. Examining practical feasibility of amorphous
curcumin-chitosan nanoparticle complex as solubility enhancement strategy
of curcumin: scaled-up production, dry powder transformation, and long-
term physical stability. Colloid Surface Physicochem Eng Aspect. 2018;537:36-
43.
17. Fonte P, Reis S, Sarmento B. Facts and evidences on the lyophilization of
polymeric nanoparticles for drug delivery. J Control Release. 2016;225:75-86.
18. Franks F. Freeze drying: from empiricism to predictability. Cryo Lett. 1990;11:
93-110.
19. Pikal M, Shah S, Roy M, Putman R. The secondary drying stage of freeze
drying: drying kinetics as a function of temperature and chamber pressure. Int
J Pharm. 1990;60(3):203-207.
20. Williams N, Polli G. The lyophilization of pharmaceuticals: a literature review.
PDA J Pharm Sci Technol. 1984;38(2):48-60.
21. Tang XC, Pikal MJ. Design of freeze-drying processes for pharmaceuticals:
practical advice. Pharm Res. 2004;21(2):191-200.
22. Bejrapha P, Surassmo S, Choi MJ, Nakagawa K, Min S-G. Studies on the role of
gelatin as a cryo-and lyo-protectant in the stability of capsicum oleoresin
nanocapsules in gelatin matrix. J Food Eng. 2011;105(2):320-331.
23. Sadikoglu H, Ozdemir M, Seker M. Freeze-drying of pharmaceutical products:
research and development needs. Dry Technol. 2006;24(7):849-861.
24. Andreani T, et al, Kiill CP, de Souza ALR. Effect of cryoprotectants on the
reconstitution of silica nanoparticles produced by solegel technology. J Therm
Anal Calorim. 2015;120(1):1001-1007.
25. Allison SD, dC Molina M, Anchordoquy TJ. Stabilization of lipid/DNA com-
plexes during the freezing step of the lyophilization process: the particle
isolation hypothesis. Biochim Biophys Acta Biomembr. 2000;1468(1-2):127-
138.
3246 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
26. Crowe LM, Reid DS, Crowe JH. Is trehalose special for preserving dry bio-
materials? Biophys J. 1996;71(4):2087-2093.
27. Almalik A, Alradwan I, Kalam MA, Alshamsan A. Effect of cryoprotection on
particle size stability and preservation of chitosan nanoparticles with and
without hyaluronate or alginate coating. Saudi Pharm J. 2017;25(6):861-867.
28. Lee MK, Kim MY, Kim S, Lee J. Cryoprotectants for freeze drying of drug nano-
suspensions: effect of freezing rate. J Pharm Sci. 2009;98(12):4808-4817.
29. Abdelwahed W, Degobert G, Fessi H. Investigation of nanocapsules stabili-
zation by amorphous excipients during freeze-drying and storage. Eur J Pharm
Biopharm. 2006;63(2):87-94.
30. Kamiya S, Nozawa Y, Miyagishima A, Kurita T, Sadzuka Y, Sonobe T. Physical
characteristics of freeze-dried griseofulvin-lipids nanoparticles. Chem Pharm
Bull. 2006;54(2):181-184.
31. Umerska A, Paluch KJ, Santos-Martinez MJ, Corrigan OI, Medina C, Tajber L.
Freeze drying of polyelectrolyte complex nanoparticles: effect of nanoparticle
composition and cryoprotectant selection. Int J Pharm. 2018;552(1e2):27-38.
32. Gokce Y, Cengiz B, Yildiz N, Calimli A, Aktas Z. Ultrasonication of chitosan
nanoparticle suspension: influence on particle size. Colloid Surface Phys-
icochem Eng Aspect. 2014;462:75-81.
33. Müller RH, Gohla S, Keck CM. State of the art of nanocrystalsespecial features,
production, nanotoxicology aspects and intracellular delivery. Eur J Pharm
Biopharm. 2011;78(1):1-9.
34. Du J, et al, Li X, Zhao H. Nanosuspensions of poorly water-soluble drugs
prepared by bottom-up technologies. Int J Pharm. 2015;495(2):738-749.
35. Konan YN, Gurny R, Alle
mann E. Preparation and characterization of sterile
and freeze-dried sub-200 nm nanoparticles. Int J Pharm. 2002;233(1-2):239-
252.
36. Franks F. Freeze-drying of bioproducts: putting principles into practice. Eur J
Pharm Biopharm. 1998;45(3):221-229.
37. De Jaeghere F, Alle
mann E, Leroux JC, et al. Formulation and lyoprotection of
poly (lactic acid-co-ethylene oxide) nanoparticles: influence on physical sta-
bility and in vitro cell uptake. Pharm Res. 1999;16(6):859-866.
38. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nano-
particles: formulation, process and storage considerations. Adv Drug Deliv Rev.
2006;58(15):1688-1713.
39. Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol
associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their
physical properties and cellular uptake. J Control Release. 2002;82(1):105-114.
40. Galindo-Rodriguez S, Allemann E, Fessi H, Doelker E. Physicochemical pa-
rameters associated with nanoparticle formation in the salting-out, emulsi-
fication-diffusion, and nanoprecipitation methods. Pharm Res. 2004;21(8):
1428-1439.
41. Molpeceres J, Aberturas M, Chacon M, Berges L, Guzman M. Stability of
cyclosporine-loaded poly-X-caprolactone nanoparticles. J Microencapsul.
1997;14(6):777-787.
42. Beirowski J, Inghelbrecht S, Arien A, van Assche I, Gieseler H. Stabilization of
nanosuspensions during freeze-drying: the role of vitrification (Part 1). In:
Proceedings of the Seventh World Meeting on Pharmaceutics. Biopharmaceutics
and Pharmaceutical Technology; 2010.
43. Beirowski J, Inghelbrecht S, Arien A, Gieseler H. Freeze-drying of nano-
suspensions, 1: freezing rate versus formulation design as critical factors to
preserve the original particle size distribution. J Pharm Sci. 2011;100(5):1958-
1968.
44. Beirowski J, Inghelbrecht S, Arien A, Gieseler H. Freeze drying of nano-
suspensions, 2: the role of the critical formulation temperature on stability of
drug nanosuspensions and its practical implication on process design. J Pharm
Sci. 2011;100(10):4471-4481.
45. Wang L, et al, Ma Y, Gu Y. Cryoprotectant choice and analyses of freeze-drying
drug suspension of nanoparticles with functional stabilisers. J Microencapsul.
2018;35(3):241-248.
46. De Jaeghere F, Alle
mann E, Feijen J, Kissel T, Doelker E, Gurny R. Freeze-drying
and lyopreservation of diblock and triblock poly (lactic acid)epoly (ethylene
oxide)(PLAePEO) copolymer nanoparticles. Pharm Dev Technol. 2000;5(4):
473-483.
47. Franks F. Freeze-drying: from empiricism to predictability. The significance of
glass transitions. Dev Biol Stand. 1992;74:9-18. discussion 19.
48. Kasper JC, Friess W. The freezing step in lyophilization: physico-chemical
fundamentals, freezing methods and consequences on process performance
and quality attributes of biopharmaceuticals. Eur J Pharm Biopharm.
2011;78(2):248-263.
49. Cui Z, Hsu C-H, Mumper RJ. Physical characterization and macrophage cell
uptake of mannan-coated nanoparticles. Drug Dev Ind Pharm. 2003;29(6):
689-700.
50. Abdelwahed W, Degobert G, Fessi H. A pilot study of freeze drying of poly
(epsilon-caprolactone) nanocapsules stabilized by poly (vinyl alcohol):
formulation and process optimization. Int J Pharm. 2006;309(1-2):178-
188.
51. Chang BS, Patro SY. Freeze-drying process development for protein pharma-
ceuticals. In: Lyophilization of Biopharmaceuticals. 2. 2004:113.
52. Chen H, Khemtong C, Yang X, Chang X, Gao J. Nanonization strategies for
poorly water-soluble drugs. Drug Discov Today. 2011;16(7e8):354-360.
53. Müller RH, Peters K. Nanosuspensions for the formulation of poorly soluble
drugs: I. Preparation by a size-reduction technique. Int J Pharm. 1998;160(2):
229-237.
54. Merisko-Liversidge E, Liversidge GG, Cooper ER. Nanosizing: a formulation
approach for poorly-water-soluble compounds. Eur J Pharm Sci. 2003;18(2):
113-120.
55. Keck CM, Müller RH. Biopharmaceutics, drug nanocrystals of poorly soluble
drugs produced by high pressure homogenisation. Eur J Pharm Biopharm.
2006;62(1):3-16.
56. Sarkari M, Brown J, Chen X, Swinnea S, Williams III RO, Johnston KP. Enhanced
drug dissolution using evaporative precipitation into aqueous solution. Int J
Pharm. 2002;243(1e2):17-31.
57. Shekunov BY, Chattopadhyay P, Seitzinger J, Huff R. Nanoparticles of poorly
water-soluble drugs prepared by supercritical fluid extraction of emulsions.
Pharm Res. 2006;23(1):196-204.
58. Kipp J. The role of solid nanoparticle technology in the parenteral delivery of
poorly water-soluble drugs. Int J Pharm. 2004;284(1-2):109-122.
59. Blagden N, de Matas M, Gavan PT, York P. Crystal engineering of active
pharmaceutical ingredients to improve solubility and dissolution rates. Adv
Drug Deliv Rev. 2007;59(7):617-630.
60. Fischer HC, Chan WC. Nanotoxicity: the growing need for in vivo study. Curr
Opin Biotechnol. 2007;18(6):565-571.
61. Rabinow BE. Nanosuspensions in drug delivery. Nat Rev Drug Discov.
2004;3(9):785.
62. Van Eerdenbrugh B, Van den Mooter G, Augustijns P. Top-down production of
drug nanocrystals: nanosuspension stabilization, miniaturization and trans-
formation into solid products. Int J Pharm. 2008;364(1):64-75.
63. Patravale V, Date AA, Kulkarni RM. Nanosuspensions: a promising drug de-
livery strategy. J Pharm Pharmacol. 2004;56(7):827-840.
64. de Waard H, Grasmeijer N, Hinrichs WL, Eissens AC, Pfaffenbach PP, Frijlink
HW. Preparation of drug nanocrystals by controlled crystallization: applica-
tion of a 3-way nozzle to prevent premature crystallization for large scale
production. Eur J Pharm Sci. 2009;38(3):224-229.
65. Sinha B, Müller RH, Mo €schwitzer JP. Bottom-up approaches for preparing
drug nanocrystals: formulations and factors affecting particle size. Int J Pharm.
2013;453(1):126-141.
66. Lee J. Drug nano-and microparticles processed into solid dosage forms:
Physical Properties. J Pharm Sci. 2003;92(10):2057-2068.
67. Gedde UW. Polymer Physics. Springer Science & Business Media; 2013.
68. Lee J, Cheng Y. Critical freezing rate in freeze drying nanocrystal dispersions.
J Control Release. 2006;111(1-2):185-192.
69. Lee J, Cheng Y. Critical freezing rate in freeze drying nanocrystal dispersions.
J Control Release. 2006;111(1-2):185-192.
70. De Waard H, Hinrichs W, Frijlink H. A novel bottomeup process to produce
drug nanocrystals: controlled crystallization during freeze-drying. J Control
Release. 2008;128(2):179-183.
71. Ma Y-Q, Zhang ZZ, Li G, Zhang J, Xiao HY, Li XF. Solidification drug nano-
suspensions into nanocrystals by freeze-drying: a case study with urso-
deoxycholic acid. Pharm Dev Technol. 2016;21(2):180-188.
72. Yue P, Wang C, Dan J, Liu W, Wu Z, Yang M. The importance of solidification
stress on the redispersibility of solid nanocrystals loaded with harmine. Int J
Pharm. 2015;480(1e2):107-115.
73. Iurian S, Bogdan C, Tomuța  L, et al. Development of oral lyophilisates con-
taining meloxicam nanocrystals using QbD approach. Eur J Pharm Sci.
2017;104:356-365.
74. Torrado S, Torrado S. Characterization of physical state of mannitol after
freeze-drying: effect of acetylsalicylic acid as a second crystalline cosolute.
Chem Pharm Bull. 2002;50(5):567-570.
75. Mehta M, Bhardwaj SP, Suryanarayanan R. Controlling the physical form of
mannitol in freeze-dried systems. Eur J Pharm Biopharm. 2013;85(2):207-213.
76. Iurian S, Tomuta I, Bogdan C, et al. Defining the design space for freeze-dried
orodispersible tablets with meloxicam. Drug Dev Ind Pharm. 2016;42(12):
1977-1989.
77. Wang Y, Zheng Y, Zhang L, Wang Q, Zhang D. Stability of nanosuspensions in
drug delivery. J Control Release. 2013;172(3):1126-1141.
78. Wang B, Zhang W, Zhang W, Mujumdar AS, Huang L. Progress in drying
technology for nanomaterials. Dry Technol. 2005;23(1e2):7-32.
79. Saez A, Guzman M, Molpeceres J, Aberturas M. Freeze-drying of poly-
caprolactone and poly (D, L-lactic-glycolic) nanoparticles induce minor par-
ticle size changes affecting the oral pharmacokinetics of loaded drugs. Eur J
Pharm Biopharm. 2000;50(3):379-387.
80. Ma J, Yang Y, Sun Y, Sun J. Optimization, Characterization and in Vitro/vivo
Evaluation of Azilsartan. .; 2016.
81. Thakkar S, Sharma D, Misra M. Comparative evaluation of electrospraying and
lyophilization techniques on solid state properties of Erlotinib nanocrystals:
assessment of In-vitro cytotoxicity. Eur J Pharm Sci. 2018;111:257-269.
82. Han J, Zhou C, Wu Y, Liu F, Wu Q. Self-assembling behavior of cellulose
nanoparticles during freeze-drying: effect of suspension concentration, par-
ticle size, crystal structure, and surface charge. Biomacromolecules.
2013;14(5):1529-1540.
83. Wolfe J, Bryant G. Freezing, drying, and/or vitrification of mem-
braneesoluteewater systems. Cryobiology. 1999;39(2):103-129.
84. Salazar J, Ghanem A, Müller RH, Mo €schwitzer JP. Nanocrystals: comparison of
the size reduction effectiveness of a novel combinative method with con-
ventional top-down approaches. Eur J Pharm Biopharm. 2012;81(1):82-90.
85. Teagarden DL, Baker DS. Practical aspects of lyophilization using non-aqueous
co-solvent systems. Eur J Pharm Sci. 2002;15(2):115-133.
 M. Mohammady et al. / Journal of Pharmaceutical Sciences 109 (2020) 3235-3247
86. Mason TG, Wilking JN, Meleson K, Chang CB, Graves SM. Nanoemulsions:
formation, structure, and physical properties. J Phys Condens Matter.
2006;18(41):R635.
87. Aboofazeli R. Nanometric-scaled emulsions (nanoemulsions). Iran J Pharm Res.
2010;9(4):325.
88. Taylor KM, Aulton ME. Aultons Pharmaceutics: The Design and Manufacture of
Medicines. Elsevier; 2013.
89. McClements DJ. Nanoemulsions versus microemulsions: terminology, differ-
ences, and similarities. Soft Matter. 2012;8(6):1719-1729.
90. Zhang Y, Shang Z, Gao C, et al. Nanoemulsion for solubilization, stabilization,
and in vitro release of pterostilbene for oral delivery. AAPS PharmSciTech.
2014;15(4):1000-1008.
91. Capek I. Degradation of kinetically-stable o/w emulsions. Adv Colloid Interface
Sci. 2004;107(2-3):125-155.
92. Gupta S, Chavhan S, Sawant KK. Self-nanoemulsifying drug delivery system
for adefovir dipivoxil: design, characterization, in vitro and ex vivo evaluation.
Colloid Surface Physicochem Eng Aspect. 2011;392(1):145-155.
93. Komatsu H, Saito H, Okada S, Tanaka M, Egashira M, Handa T. Effects of the
acyl chain composition of phosphatidylcholines on the stability of freeze-
dried small liposomes in the presence of maltose. Chem Phys Lipids.
2001;113(1e2):29-39.
94. McClements D, Decker E, Weiss J. Emulsion-based delivery systems for lipo-
philic bioactive components. J Food Sci. 2007;72(8):R109-R124.
95. Hedoux A, Paccou L, Achir S, Guinet Y. Mechanism of protein stabilization by
trehalose during freeze-drying analyzed by in situ micro-Raman spectros-
copy. J Pharm Sci. 2013;102(8):2484-2494.
96. do Vale Morais AR, do Nascimento Alencar E,
Júnior FHX, et al. Freeze-drying
of emulsified systems: a review. Int J Pharm. 2016;503(1e2):102-114.
97. Li F, Wang T, He HB, Tang X. The properties of bufadienolides-loaded nano-
emulsion and submicro-emulsion during lyophilization. Int J Pharm.
2008;349(1e2):291-299.
98. Wei W, Mo C, Guohua C. Issues in freeze drying of aqueous solutions. Chin J
Chem Eng. 2012;20(3):551-559.
99. Couvreur P, Barratt G, Fattal E, Vauthier C. Nanocapsule technology: a review.
Crit Rev Ther Drug Carrier Syst. 2002;19(2).
100. Anton N, Benoit J-P, Saulnier P. Design and production of nanoparticles
formulated from nano-emulsion templatesda review. J Control Release.
2008;128(3):185-199.
101. Letchford K, Burt H. A review of the formation and classification of amphi-
philic block copolymer nanoparticulate structures: micelles, nanospheres,
nanocapsules and polymersomes. Eur J Pharm Biopharm. 2007;65(3):259-269.
102. Nakagawa K, Surassmo S, Min SG, Choi MJ. Dispersibility of freeze-dried poly
(epsilon-caprolactone) nanocapsules stabilized by gelatin and the effect of
freezing. J Food Eng. 2011;102(2):177-188.
103. Bejrapha P, Min SG, Surassmo S, Choi MJ. Physicothermal properties of freeze-
dried fish oil nanocapsules frozen under different conditions. Dry Technol.
2010;28(4):481-489.
104. Searles JA, Carpenter JF, Randolph TW. Annealing to optimize the primary
drying rate, reduce freezing-induced drying rate heterogeneity, and deter-
mine Tg' in pharmaceutical lyophilization. J Pharm Sci. 2001;90(7):872-887.
105. Abdelwahed W, Degobert G, Fessi H. Freeze-drying of nanocapsules: impact
of annealing on the drying process. Int J Pharm. 2006;324(1):74-82.
106. Bexiga NM, Bloise A, Alencar AM, Stephano MA. Freeze-drying of ovalbumin-
loaded carboxymethyl chitosan nanocapsules: impact of freezing and
annealing procedures on physicochemical properties of the formulation
during dried storage. Dry Technol. 2018;36(4):400-417.
107. Kamiya S, Kurita T, Miyagishima A, Itai S, Arakawa M. Physical properties of
griseofulvin-lipid nanoparticles in suspension and their novel interaction
mechanism with saccharide during freeze-drying. Eur J Pharm Biopharm.
2010;74(3):461-466.



108. Curi c A, Keller BL, Reul R, Mo €schwitzer J, Fricker G. Development and
lyophilization of itraconazole loaded poly (butyl cyanoacrylate) nanospheres
as a drug delivery system. Eur J Pharm Sci. 2015;78:121-131.
109. de Chasteigner S, Cave
G, Fessi H, Devissaguet JP, Puisieux F. Freeze-drying of
itraconazole-loaded nanosphere suspensions: a feasibility study. Drug Dev
Res. 1996;38(2):116-124.
110. He W, Lu Y, Qi J, Chen L, Hu F, Wu W. Nanoemulsion-templated shell-cross-
linked nanocapsules as drug delivery systems. Int J Pharm. 2013;445(1e2):69-
78.
111. Lozano M, Esteban H, Brea J, Loza M, Torres D, Alonso M. Intracellular delivery
of docetaxel using freeze-dried polysaccharide nanocapsules. J Microencapsul.
2013;30(2):181-188.
112. Oyarzun-Ampuero FA, Rivera-Rodríguez GR, Alonso MJ, Torres D. Hyaluronan
nanocapsules as a new vehicle for intracellular drug delivery. Eur J Pharm Sci.
2013;49(4):483-490.
113. Allu S, Bolla G, Tothadi S, Nangia A. Supramolecular synthons in bumetanide
cocrystals and ternary products. Cryst Growth Des. 2017;17(8):4225-4236.
114. Desiraju GR. Supramolecular synthons in crystal engineeringda new organic
synthesis. Angew Chem. 1995;34(21):2311-2327.
115. Zhao L, Raval V, Briggs NE, et al. From discovery to scale-up: a-lipoic acid:
nicotinamide co-crystals in a continuous oscillatory baffled crystalliser.
CrystEngComm. 2014;16(26):5769-5780.
116. Rodrigues M, Baptista B, Lopes JA, Sarraguça MC. Pharmaceutical cocrystalli-
zation techniques. Advances and challenges. Int J Pharm. 2018;547(1e2):404-
420.
3247
117. Eddleston MD, Patel B, Day GM, Jones W. Cocrystallization by freeze-drying:
preparation of novel multicomponent crystal forms. Cryst Growth Des.
2013;13(10):4599-4606.
118. Grossjohann C, et al, Serrano DR, Paluch KJ. Polymorphism in sulfadimidine/4-
aminosalicylic acid cocrystals: solid-state characterization and physico-
chemical properties. J Pharm Sci. 2015;104(4):1385-1398.
119. Zhang D, Tan T, Gao L, Zhao W, Wang P. Preparation of azithromycin nano-
suspensions by high pressure homogenization and its physicochemical
characteristics studies. Drug Dev Ind Pharm. 2007;33(5):569-575.
120. Liu D, Xu H, Tian B, et al. Fabrication of carvedilol nanosuspensions through
the anti-solvent precipitationeultrasonication method for the improvement
of dissolution rate and oral bioavailability. AAPS Pharm Sci Tech. 2012;13(1):
295-304.
121. Van Eerdenbrugh B, Froyen L, Martens J, et al. Characterization of physico-
chemical properties and pharmaceutical performance of sucrose co-
freezeedried solid nanoparticulate powders of the anti-HIV agent loviride
prepared by media milling. Int J Pharm. 2007;338(1e2):198-206.
122. Ibrahim AH, Rosqvist E, Smått J-H, et al. Formulation and optimization of
lyophilized nanosuspension tablets to improve the physicochemical properties
and provide immediate release of silymarin. Int J Pharm. 2019;563:217-227.
123. Kanthamneni N, Valiveti S, Patel M, Xia H, Tseng YC. Enhanced bioavailability
of danazol nanosuspensions by wet milling and high-pressure homogeniza-
tion. Int J Pharm Investig. 2016;6(4):218.
124. Oktay AN, Karakucuk A, Ilbasmis-Tamer S, Celebi N. Dermal flurbiprofen
nanosuspensions: Optimization with design of experiment approach and in
vitro evaluation. Eur J Pharm Sci. 2018;122:254-263.
125. Lyu J, Zhang H, Wang L, Gao J. Preparation and Dissolution of Loratadine
Nanosuspension Lyophilized Powder. China Pharmacist. 2018;21(5):809-813.
126. He J, Han Y, Xu G, et al. Preparation and evaluation of celecoxib nanosuspensions
for bioavailability enhancement. RSC Adv. 2017;7(22):13053-13064.
127. Rao YM, Kumar MP, Apte S. Formulation of nanosuspensions of albendazole
for oral administration. Curr Nanosci. 2008;4(1):53-58.
128. Liversidge GG, Cundy KC. Particle size reduction for improvement of oral
bioavailability of hydrophobic drugs: I. Absolute oral bioavailability of
nanocrystalline danazol in beagle dogs. Int J Pharm. 1995;125(1):91-97.
129. Ain-Ai A, Gupta PK. Effect of arginine hydrochloride and hydroxypropyl cellulose
as stabilizers on the physical stability of high drug loading nanosuspensions of a
poorly soluble compound. Int J Pharm. 2008;351(1e2):282-288.
130. Lou H, Zhang X, Gao L, et al. In vitro and in vivo antitumor activity of oridonin
nanosuspension. Int J Pharm. 2009;379(1):181-186.
131. Teeranachaideekul V, Junyaprasert VB, Souto EB, Müller RH. Development of
ascorbyl palmitate nanocrystals applying the nanosuspension technology. Int
J Pharm. 2008;354(1e2):227-234.
132. de Waard H, De Beer T, Hinrichs WL, Vervaet C, Remon JP, Frijlink HW.
Controlled crystallization of the lipophilic drug fenofibrate during freeze-
drying: Elucidation of the mechanism by in-line Raman spectroscopy. AAPS J.
2010;12(4):569-575.
133. Ma YQ, Li G, Xu JH, et al. Combination of submicroemulsion and phospholipid
complex for novel delivery of ursodeoxycholic acid. Pharm Dev Technol.
2014;19(3):363-372.
134. Gol D, Thakkar S, Misra M. Nanocrystal-based drug delivery system of ris-
peridone: lyophilization and characterization. Drug Dev Ind Pharm.
2018;44(9):1458-1466.
135. Khayata N, Abdelwahed W, Chehna MF, Charcosset C, Fessi H. Preparation of
vitamin E loaded nanocapsules by the nanoprecipitation method: From lab-
oratory scale to large scale using a membrane contactor. Int J Pharm.
2012;423(2):419-427.
136. Torrecilla D, Lozano MV, Lallana E, et al. Anti-tumor efficacy of chitosan-g-
poly (ethylene glycol) nanocapsules containing docetaxel: anti-TMEFF-2
functionalized nanocapsules vs. non-functionalized nanocapsules. Eur J Pharm
Biopharm. 2013;83(3):330-337.
137. Surassmo S, Min SG, Bejrapha P, Choi MJ. Effects of surfactants on the physical
properties of capsicum oleoresin-loaded nanocapsules formulated through
the emulsionediffusion method. Food Res Int. 2010;43(1):8-17.
138. Lu B, Xiong S-B, Yang H, Yin X-D, Zhao RB. Mitoxantrone-loaded BSA nano-
spheres and chitosan nanospheres for local injection against breast cancer
and its lymph node metastases: I: Formulation and in vitro characterization.
Int J Pharm. 2006;307(2):168-174.
139. Pfeifer BA, Burdick JA, Langer R. Formulation and surface modification of poly
(ester-anhydride) micro-and nanospheres. Biomaterials. 2005;26(2):117-124.
140. Donini C, Robinson D, Colombo P, Giordano F, Peppas N. Preparation of poly
(methacrylic acid-g-poly (ethylene glycol)) nanospheres from methacrylic
monomers for pharmaceutical applications. Int J Pharm. 2002;245(1e2):83-
91.
141. Wagh P, Mujumdar A, Naik JB. Preparation and characterization of ketorolac
tromethamine-loaded ethyl cellulose micro-/nanospheres using different
techniques. Particul Sci Technol. 2019;37(3):347-357.
142. ElShagea HN, ElKasabgy NA, Fahmy RH, Basalious EB. Freeze-Dried Self-
Nanoemulsifying Self-Nanosuspension (SNESNS): a New Approach for the
Preparation of a Highly Drug-Loaded Dosage Form. AAPS Pharm Sci Tech.
2019;20(7):258.
143. Wang L, Zhao X, Zu Y, et al. Enhanced dissolution rate and oral bioavailability
of ginkgo biloba extract by preparing nanoparticles via emulsion solvent
evaporation combined with freeze drying (ESE-FR). Rsc Adv. 2016;6(81):
77346-77357.