Review

Received: 2 June 2010 Revised: 15 July 2010 Accepted: 23 July 2010 Published online in Wiley Online Library: 2 September 2010

(wileyonlinelibrary.com) DOI 10.1002/jctb.2495

Controlled freezing and freeze drying:

a versatile route for porous and
micro-/nano-structured materials†
Lei Qian and Haifei Zhang∗

Abstract
Freeze drying is a process whereby solutions are frozen in a cold bath and then the frozen solvents are removed via sublimation
under vacuum, leading to formation of porous structures. Pore size, pore volume and pore morphology are dependent on
variables such as freeze temperature, solution concentration, nature of solvent and solute, and the control of the freeze
direction. Aqueous solutions, organic solutions, colloidal suspensions, and supercritical CO2 solutions have been investigated
to produce a wide range of porous and particulate structures. Emulsions have recently been employed in the freeze drying
process, which can exert a systematic control on pore morphology and pore volume and can also lead to the preparation
of organic micro- and nano-particles. Spray freezing and directional freezing have been developed to form porous particles
and aligned porous materials. This review describes the principles, latest progress and applications of materials prepared by
controlled freezing and freeze drying. First of all the basics of freeze drying and the theory of freezing are discussed. Then the
materials fabricated by controlled freezing and freeze drying are reviewed based on their morphologies: porous structures,
microwires and nanowires, and microparticles and nanoparticles. The review concludes with new developments in this area
and a brief look into the future.

c 2010 Society of Chemical Industry

Keywords: controlled freezing; porous materials; porous ceramics; aligned porous structures; microwires; nanofibres; organic
nanoparticles; porous microparticles

INTRODUCTION Monte and co-worker5 dealt with porous polymers/composites

Freeze drying, also known as lyophilization, has been widely
used to prepare porous materials for tissue engineering and
biological applications,1,2 and also used in the pharmaceutical
industry to improve the stability of labile drugs.3 In recent years,
the method of freeze drying has been explored as a unique route
to produce novel porous materials. Different types of porous
materials such as aligned porous materials and hybrid porous
materials, have been successfully prepared by freeze drying.4 – 6
Other processes for the preparation of porous materials include:
leaching of particles; emulsion templating; phase separation;
three-dimensional (3D) printing and electrochemistry.7 – 14 In
general, these methods require the use of large amounts of
organic solvents and a lengthy washing or etching procedure. A
freeze drying process can provide certain advantages. For example,
aqueous solutions are often used to prepare porous materials by
freeze drying. Water is an environment-friendly solvent and the
use of ice crystals as porogens is green and sustainable. This is
particularly beneficial for biological applications. When removing
the solvent, the freeze drying process does not bring impurities
into the samples and a further purifying process is therefore
not necessary. More importantly, by changing variables during
freezing, it is possible to produce materials with a wealth of
pore morphologies and nanostructures. It is believed that such
materials can be used in a wide range of applications such as
bioengineering, drug delivery, catalyst support and separation.
Two excellent reviews on ice templating were published in 2008,
172
and porous ceramics. In addition in a report on progress on the
fracture toughness of advanced materials Launey et al. explained
the design of high toughness bioinspired structural materials by
freezing.16 There have been some significant developments in the
preparation of materials using the freezing approach, for example,
organic solutions for biocompatible scaffolds; combination of
emulsions and freezing for porous materials and nanoparticles,
microwires/nanofibres by the controlled freezing of colloidal
suspensions and dilute polymer solutions. These topics and also
spray freeze drying for microparticles are covered in this review.
This review first of all explains the basics of freeze drying
methods and the theory of freezing, then introduces the materials
obtained using the techniques based on their structures (i.e.
porous structures, microwires and nanowires, microparticles and
nanoparticles). Because there is a significant amount of work to
cover for porous structures, the materials are discussed based on
the systems used for their preparation; aqueous solutions, colloidal
suspensions, organic solutions and emulsions. Although porous
∗ Correspondence to: Haifei Zhang, Department of Chemistry, University of
Liverpool, Crown Street, Liverpool, L69 7ZD, UK. E-mail: zhanghf@liv.ac.uk
† This paper was presented at The 2009 Annual Meeting of The Chinese Society of
Chemical Science and Technology in the UK (CSCST) & The Society of Chemical
Industry – Chinese UK Section (SCI – CS).
Department of Chemistry, University of Liverpool, Crown Street, Liverpool, L69

one by Deville15 covered porous ceramics and another by del 7ZD, UK

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materials have been prepared conventionally by immersing the

liquid samples in liquid nitrogen, recent development in materials
synthesis has focused on the use of directional freezing for the
preparation of aligned or layered porous structures. Under each
system of preparation, initially we introduce the structures of the
porous materials generated by conventional freezing and then the
structures obtained using directional freezing.

BASIS OF FREEZE DRYING

A freeze drying process consists of three stages: freezing, primary
drying and secondary drying.3,17,18 The freezing process is realized
by contacting a liquid sample with or placing it in a cold bath.
The frozen sample is then placed in a freeze dryer to remove the
Figure 1. Schematic representation of the directional freezing process.
frozen solvent by sublimation. During the freeze drying process,
Ice crystals grow in one direction and the solutes (such as particles,
the frozen sample should be kept below the glass transition polymeric molecules or mixture of them) are excluded and solidified
temperature or melting point and the frozen solvent is removed between the crystals.4 (Reprinted with permission from Ref. 4, copyright
under vacuum.17,18 Porous structures are formed from the voids Wiley Periodicals 2007).
left by the removal of the solvent. Thus, the frozen solvent acts as
porogen to produce porous materials.

Controlled freezing
The freezing step is very important in order to produce desirable
porous structures. During the freezing step, solvent crystals grow
and solute molecules are excluded from the frozen solvent until
the sample is completely frozen. Different conditions of freezing
including the freezing temperature, solute concentration, solvent
type, and direction of freezing can have a great impact on
the resulting pore structure of the materials. When an aqueous
solution is frozen in liquid nitrogen, the extremely low temperature
( −196 ◦ C) of liquid nitrogen results in rapid formation of ice nuclei
and the growth of small ice crystals. However, when the freezing
process is carried out at a higher temperature (e.g. −20 ◦ C), ice
nucleation is slow and the nuclei tend to grow into larger ice
crystals, which leads to the preparation of materials with large and
random pores after the freeze drying process Figure 2. Schematic representation of a SFL process using liquid nitrogen
as the cryogenic medium.19 (Reprinted with permission from Ref. 19,
The direction of freezing also has a major effect on pore copyright Elsevier Science 2002).
morphology,4 by controlling the direction of freezing the growth
of ice crystal can be orientated in one direction, a process called
directional freezing (Fig. 1). This process is realized by applying a sample volume and exposed surface area, and product resistance.3
high temperature gradient across the sample with the ice crystals Secondary drying is carried out to desorb the unfrozen solvent
growing from the low to the high temperature end. Then removal bound to the polymer and for this process a lower vacuum level
of orientated ice crystals by freeze drying generates materials with than for primary drying is used to remove the bound water.
unidirectional pores.5,6 This secondary drying process may be particularly important in
Spray-freezing into liquid (SFL) is a particle engineering technol- pharmaceutical applications.3
ogy where the feed solution is atomized beneath the surface of
a cryogenic liquid (e.g. liquid nitrogen) at a high constant pres-
sure (e.g. 5000 psi) maintained by a HPLC pump (Fig. 2).19 The THEORY OF FREEZING
impingement of the feed solution and the cryogenic liquid results
When an aqueous solution is frozen, ice crystals grow and solute
in intense atomization of the feed into microdroplets, which in-
molecules are excluded by these crystals. Because impurities have
stantly freeze in the cryogen. The frozen microparticles are then
a very low solubility in ice crystals, a concentration gradient
separated and subjected to a freeze drying process to produce dry
of the solute is formed and the solute concentration ahead
microparticles.
of the ice front is increased.20 This increase of concentration
reduces the melting point of the solution, which results in the
Drying process formation of a constitutional supercooling zone and finally this
The drying process is usually carried out in a freeze drier with can break down the planar interface, and the ice cell grows.21 – 23
temperature-controlled shelves. Primary drying occurs as the This phenomenon is called Mullins–Sekerka instability, according
frozen solvent sublimes on reduction of the pressure to a value to which, the primary ice-template structure is dependent on
below the triple point. This step is usually the most time-consuming the destabilizing solute interfacial concentration gradient and
step in the process directly related to the ice sublimation rate and is the surface energy that opposes cell formation. The following
173

determined by factors such as level of vacuum, shelf temperature, equation is obtained by linear stability analysis for the growth rate

J Chem Technol Biotechnol 2011; 86: 172–184

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 www.soci.org
of a perturbation (δ) to the planar interface, of magnitude δ̇ as a
function of the instability.6,21 – 23
    
 −2Tmω2 ω∗ − V (1 − k) − 2G ω∗ − V (1 − k) 
D D
Vω  
 +2mG ω ∗− V 
δ̇ c D
=
δ ks − kl V
2G ω∗ − (1 − k) + 2ωmGc
ks + kl D
1/2
V V
ω∗ = + ( )2 + ω2
2D 2D
where ω is the frequency and m is the slope of the liquidus
line on the phase diagram, V is the growth velocity, Tm is the
equilibrium melting point,  is the ratio of the surface energy
and the latent heat of fusion, D is the diffusion coefficient, k is
the partition coefficient, G is the temperature gradient, GC is the
solute concentration gradient at the interface and ks and kl are the
thermal conductivities of the ice and solution phases. This equation
was used to predict the pore spacings of aligned porous poly(vinyl
alcohol) (PVA) formed by directional freezing. The predicted value
and the experimental value agreed reasonably well.6
For a multicomponent system including molecules and colloids,
the analysis is much more complicated. To make it simple a
theoretical study of the freezing process was conducted based
on freezing a suspension of hard spheres.24 – 26 A cryomicroscope
was used to determine critical interface velocities marking the
transition between repulsion and entrapment of spherical latex
particles by an advancing ice–liquid interface.25 For a particle
moving very close to a freezing front, a repulsive force between
the particle and the frozen phase is derived as:
a0 n
Fσ = 2π Rσ ( ) The spatial spreading and proliferation of endothelial cells and
d mouse embryonic fibroblasts was investigated on porous chitosan
where R is the particle radius, a0 is the average intermolecular
distance, d is the distance between the particle and the ice front, n
is a parameter with a value ranged from 1 to 4, σ is the balance of
the surface forces at the ice/solution/particle boundary. The ( a0 )n
d
term is a correction to the disjoining force on the particle.
The attractive drag force is derived for the case of a flat interface:
6π ηνR2
Fη =
d
where η is the viscosity of the liquid suspension and v is the ice
growth velocity.
A balance of forces yields a critical ice growth velocity at which
particles theoretically are rejected or encapsulated by the ice:
σ d a0 n
vc = ( )
3ηr d
Recently, mathematical models have been developed by
Peppin et al.27 – 28 for the unidirectional solidification of hard-
sphere colloidal suspensions These investigations were focused
on the planar interfaces moving at low velocities (0.1–1 µm s−1 )
by optical observation of the systems, which reach equilibrium
by Brownian diffusion. To understand the critical parameters
controlling the stability of solidification interfaces in colloidal
suspensions where the freezing velocities are in the range
10–100 µm s−1 , X-ray radiography and tomography are used to
investigate the stability of a cellular interface.29 The constitutional
supercooling and interface instabilities are explained by a partial
174
Brownian diffusion.
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L Qian and H Zhang
Figure 3. SEM images of porous chitosan scaffolds prepared by freeze
drying. (A) the cylinder scaffold made from freezing 2 wt% chitosan in
liquid nitrogen; (B) the planar scaffold made from freezing 2 wt% chitosan
in a dish contacting with dry ice.32 (Reprinted with permission from Ref.
32, copyright Elsevier 1999).
POROUS STRUCTURES
Aqueous solutions
There is little doubt that water-based systems have been most
extensively investigated for the production of porous materials via
the freezing and freeze drying approach. Examples of the processes
studied include the preparation of hydrophilic porous polymeric
structures from aqueous polymer solutions with, for example,
PVA hydrogels being prepared, which showed interesting porous
structures.30,31 Chitosan, a deacetylated derivative of chitin, can
be dissolved in acidic water. In the process of preparing porous
chitosan, chitosan solutions were prepared by dissolving in
0.2 mol L−1 acetic acid and then frozen in a cylindrical glass tube
by immersion into liquid nitrogen.32 By freezing the solutions at
different temperatures, it was found that the mean pore diameter
was increased with increasing temperature of freezing (Fig. 3).
scaffolds.33 Glycosaminogylcan (an anionic polysaccharide of the
extracellular matrix) was covalently immobilized on chitosan
scaffolds to form a 3D network. These composite scaffolds were
then used for the expansion of human cord blood stem cells in
perfusion culture.34 A sodium alginate solution was mixed with
a chitosan solution and the mixture solution was then processed
to produce chitosan–alginate scaffolds.35 3D silk fibroin scaffolds
have also been prepared for the study of cell proliferation and
migration and the effects of different freezing temperature regimes
on the resulting pore microstructure were investigated.36
When the freezing process is conducted with a cold source
of higher temperature (e.g, dry ice at −78 ◦ C or a freezer at
−20 ◦ C) or the process is not well controlled, materials with
random pores or partially aligned pores can be produced. To
produce porous materials with well-controlled aligned channels,
a directional freezing process has been developed.4 – 6 This can
easily be achieved by dipping a vial containing the liquid sample
into liquid nitrogen at a controlled rate although a purpose-built
facility can have more precise control of the process.
Porous PVA with aligned porous structures was obtained by
directional freezing of 5 wt% aqueous PVA solutions and then
freeze drying, as shown in Fig. 4(A). The spacing between the
aligned walls can be tuned from 12 to 50 µm over a range of
freezing rates of 10–100 µm s−1 .6 Del Monte et al.37 investigated
the preparation of aligned porous PVA and their use for drug
delivery. Higher concentrations of PVA (7.8 and 10 wt%) with
the same molecular weight led to smaller pore sizes. The
porous PVA was used as matrix for the release of ciprofloxacin
from 10 min to several days. Composites of polypyrrole and
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Freeze drying for porous materials and particles
Figure 4. (A) Aligned porous PVA prepared by directional freezing.6 (Reprinted with permission from Ref. 6, copyright Nature Publishing Group 2005.)
(B) Aligned porous graphene-PVA composite.39 (Reprinted with permission from Ref 39, copyright Wiley Periodicals 2009).
PVA were fabricated via vapour deposition polymerization of
pyrrole onto aligned porous PVA-FeCl3 formed by directional
freezing.38 The apparent conductivity of the composite containing
20 wt% polypyrrole was approximately 0.1 S cm−1 . Homogeneous
mixtures of polystyrene sulfonate-stabilized graphene sheets and
PVA were unidirectionally frozen to fabricate graphene–polymer
nanocomposites.39 Fig. 4(B) shows the aligned porous structure of
the composite. Mechanical testing indicated that the compression
module was almost an order of magnitude higher than that of
porous PVA alone.
In principle, most hydrophilic polymers and natural polymers
may be processed to prepare both porous and aligned structures.
The polymer may be dissolved in water at room temperature or
dissolved in water under different pH conditions or at elevated
temperatures. For example, aligned porous gelatin with a tunable
pore size and high porosity up to 98% have been produced.40
In vitro cell culture studies on porous gelatin indicated that
the scaffolds were non-toxic to cartilage cells. Starch scaffolds
with uniaxial aligned channels were prepared and then used as
templates to produce hierarchically porous silica.41 The effect of
water crystallization during freezing was employed for the directed
organization of preformed β-sheet fibrils and for the production
of porous materials comprising lamella-like layers.42
Aligned porous inorganic structures may be produced by
the directional freezing of inorganic precursor sol or gel. Thus
commercial sodium silicate solutions were diluted with water and
the pH was adjusted to 3. The clear sols obtained were aged at 303 K
and then unidirectionally frozen. After aging at low temperature,
the frozen samples were washed by solvent exchange and freeze
dried to produce monolithic silica gel microhoneycombs. The
surface area of the resulting silica gel could reach 780 m2 g−1 .43
Silica gels with various pore morphologies were prepared through
directional freezing of silica hydrogels and silica hydrosols. The
dominant factor governing the pore shape of the silica gel was
found to be the mobility of the silica.44 Resorcinol-formaldehyde
(RF) hydrogels were prepared and then unidirectionally frozen.
The RF cryogels were then freeze dried after the water contained
inside the sample was replaced by t-butanol. The dry RF gel with
a microhoneycomb structure was converted to porous carbon by
pyrolyzing in an inert atmosphere.45 Macroporous monoliths of
SiO2 –Al2 O3 cryogels were prepared using sodium silicate solution
and aluminium nitrate as the Si and Al sources.46 Titania–silica
cryogels with controlled pore shapes were prepared and the use
of these cryogels for photocatalysis was demonstrated.47
The directional freezing approach could be employed to prepare
hierarchical biohybrid materials. Aqueous silica sol, PVA and an
enzyme (pig liver esterase) were mixed together to form a gel.
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By using an ice-segregation-induced self-assembly (ISISA) process,
the resulting gel was cooled to liquid nitrogen temperature and
subsequent freeze drying resulted in an aligned porous material
with micrometer-sized pores.48 The prepared biohybrid structures
contained up to six levels of space organization and could preserve
the enzyme structure and functionality.
Aqueous colloidal suspensions
Inorganic (mainly silica and metal oxides) and polymer colloidal
suspensions can be frozen and freeze dried to prepare porous
inorganic or composite materials. These colloids are used as build-
ing blocks for the fabrication of porous materials. Interconnected
porous materials or aligned/layered porous structures may be pro-
duced depending on the freezing conditions. In general, a polymer
dispersant is required to stabilize the colloids in suspension or act
as a directing agent in the freezing process. For example, PVA or
sodium carboxymethylcellulose (SCMC) was added to silica col-
loidal suspensions to produce porous materials with well-defined
parallel microchannels or layered structures.6,49 Without PVA in
the suspension, only loose silica microplates or microparticles
were produced. PVA directed the orientated growth of ice crystals
and held the silica colloids together in the porous structures.6 By
increasing the rate of ice growth and the temperature gradient
when freezing silica colloidal suspensions, Nishihara et al.50 could
reduce the channel size to 530 nm and with the addition of dex-
tran to the silica suspension, the channel size could be as small as
180 nm.
An important aspect of the research in this area is the utilization
of aqueous ceramic suspensions to fabricate advanced porous
ceramics. Slurries containing high content of ceramic powders
(e.g. 20–50 wt%) in water have been employed to produce
different types of porous ceramics. It is essential to add a
polymeric dispersant to stabilize the ceramic particles and then
to bind the particles together in the freeze dried materials.
The use of freeze drying to prepare porous ceramics is also
widely known as freeze-casting.15 Alumina is one of the widely
used ceramic powders and the pore structure and properties of
porous alumina prepared by freeze-casting have been extensively
investigated. Regular patterns were obtained in such structures
by controlling the freezing of alumina slurries followed by
ice sublimation and sintering.20 Processing variables including
composition of the suspension, freezing rate, and patterning
of the freezing surface were studied to control the resulting
architecture of the porous alumina.51 Tallón et al. investigated
the shaping of porous alumina bodies by freeze casting. The
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different parameters of the process, such as the solids content, the
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Figure 5. Influence of interfacial chemistry on the mechanical response of alumina/PMMA layered composites. (A) The strength of the hybrid composites
can reach values above 200 MPa. (B) Chemical grafting increases the adhesion at the organic/inorganic interface and enhances both strength and
initiation toughness. The SEM images show the fracture surface of lamellar material with non-grafted interfaces (C) and chemical grafting surfaces (D).
Scale bars indicate 5 µm for images (C) and (D).56 (Reprinted with permission from Ref. 56, copyright American Association for the Advancement of
Science 2008).

cryoprotector (glycerol) and the freezing conditions (temperature
and device) were studied to determine their influence on the
microstructure, density and porosity of the green and sintered
alumina bodies.52 An impregnate–freeze-casting approach was
utilized to fabricate porous alumina. A polyurethane sponge
was first impregnated with an aqueous alumina slurry and then
subjected to unidirectional solidification and freeze drying. The
use of the porous template introduced a local interruption to
the lamellar structure in alumina.53 Foamed aqueous alumina
suspensions were unidirectionally frozen to prepare alumina
ceramics with aligned porous walls. In this process, PVA was
used as a binder and emulsifying agent to stabilize air bubbles
generated in aqueous alumina suspensions.54 The freeze dried
and subsequently calcined porous alumina could be further
annealed and bridged at high temperatures to improve its
mechanical strength. Inspired by the hierarchical complexity
present in nature (e.g. the structure of nacre),16,55 Ritchie
and co-workers infiltrated annealed porous lamellar alumina
with a free radical polymerization of polymethylmethacrylate
(PMMA). Highly toughened hybrid composites were produced
with toughness nearly 300 times higher (in energy terms) than
either of the constituents.56,57 Figure 5 shows the mechanical
strength and initiation toughness of the alumina/PMMA hybrid
composite. Chemical grafting at the interface between alumina
and PMMA enhanced both strength and initiation toughness. The
morphologies of non-grafted and chemically grafted interfaces
176
could be readily translated to multiple material combinations.
Indeed, the same group further demonstrated the design and
fabrication of high-performance ceramic–metal composites with
Al2 O3 /Al-Si laminates achieving an excellent fracture toughness
of 40 MPa m1/2 at a tensile strength of approximately 300 MPa.58
Hydroxyapatite (HA) comprises the inorganic part of a number
of natural hard tissues such as bones thus HA scaffolds have been
widely used in bone tissue engineering.59,60 Aqueous suspensions
of HA powders are another type of ceramic powder that have
been studied extensively. Aqueous suspensions containing 10
vol % HA particles and 0.75 wt % dispersant were subjected to
unidirectional freezing for the preparation of porous HA. In vitro
cellular response to HA scaffolds with orientated architectures was
investigated and showed that the scaffolds could be used for bone
repairs.61 Dramatic changes in the microstructure of porous HA
was observed by freezing suspensions containing binary mixture
of solvents (water-dioxane and water-glycerol).62 HA was modified
by the addition of SiO2 nanoparticles during freeze-casting. The
addition of SiO2 introduced a partial phase transformation of
HA to β-tricalcium phosphate and improved the stability of the
structure due to less shrinkage after sintering. The impact of surface
roughness of the porous ceramics on adhesion, proliferation, and
differentiation of human osteoblast-like cells was investigated.63
To make biohybrid composites, layered porous HA was filled with
epoxy to mimic the structure of natural nacre. The hybrid materials
could be used for osseous tissue regeneration with strengths up

were revealed by SEM images. This approach was flexible and to four times higher than those of materials currently used for

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Freeze drying for porous materials and particles
Figure 6. (A) Aligned porous MWCNT with silk fibroin, inset scale bar 500 nm.72 (Reprinted with permission from Ref. 72, copyright Elsevier 2009.)
(B) Morphology of macroporous MWCNT/chitosan. The scale bar is 20 um.74 (Reprinted with permission from Ref. 74, copyright Elsevier 2008).
implantation.55 Porous composite materials of biopolymers/HA
such as collagen/HA and alginate-chitosan/HA have also been
produced. These composites showed good biocompatibility to
bone cells and exhibited potential application as scaffolds for
bone tissue engineering.64 – 66
In addition to silica and ceramic powder suspensions, aqueous
organic colloid suspensions have been used to fabricate porous
organic materials or composites. Like ceramic colloidal suspen-
sions, a polymer dispersant is normally present to stabilize the
organic colloids suspended in water, e.g. polystyrene (PS) colloids
were suspended in an aqueous SCMC solution. The mixture was
directionally frozen and freeze dried to produce layered porous
structures49 and it was found that the concentration of PS colloids
could affect the thickness of the layers.49 Graphene/PS nanocom-
posites were prepared by freeze drying a mixed suspension of
graphene and PS colloids followed by compression molding. A
maximum conductivity of about 15 S cm−1 was achieved with
1.6–2.0 wt% nanofiller.67 Oppositely charged polymer microgels
which could be suspended in water at pH = 7.0 have been
utilized to prepare aligned porous structures. Due to the oppo-
site charges of the microgels, the freeze dried materials were
not soluble in water.68 In another study, dry poly(styrene-co-
2-hydroxyethylmethacrylate) colloids (average diameter 385 nm)
were added to silica sol in a 74.2/25.8 polymer/silica volume ratio.
The resulting colloidal suspensions were then used to prepare hon-
eycomb silica with highly ordered interconnected macroporous
walls.69 It was observed that freeze drying of a waterborne mixture
of silica nanoparticles (LUDOX TM-40) and poly(vinyl laurate) la-
texes could generate nanocomposite foams70 and the addition of
a third colloidal component, carbon black particles, transformed
these into conducting nanocomposite foams.70
Due to their unique and beneficial properties, carbon nanotubes
(CNTs) have been intensively investigated as a nanostructured car-
rier or incorporated into other materials to form nanocomposites.
It is not surprising that CNTs have been included in the prepara-
tion of porous composites via an ice-templating route, for which
the CNTs are dispersed in aqueous solutions stabilized by a poly-
mer. For example, multiwalled carbon nanotubes (MWCNTs) were
dispersed in chitosan solutions and then processed to produce
conductive macroporous chitosan–CNT scaffolds. Conductivity
measurements revealed that the optimal threshold concentra-
tion of MWCNTs was 2.5 wt% (or 45 vol%) of the final scaffold
precipitate.71 MWCNT cryogels with aligned and non-aligned
porous structures could also be prepared by directional freez-
ing and flash freezing in liquid nitrogen with a minor fraction of
silk fibroin as the structure binder. The aligned structure (Fig. 6(A))
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showed better thermal stability and electrical conductivity than
the non-aligned one.72 MWCNT–chitosan scaffolds were found to
be compatible with E. coli and could be used for bacteria immo-
bilization and proliferation within the structures.73 The adhesion,
viability and proliferation of the C2C12 cell line (myoblastic mouse
cell) onto the external surface of MWCNT–chitosan scaffolds were
evaluated in vitro and quantified by MTT assays. A chamber-like
structure of the scaffold is shown in Fig. 6(B). The recombinant
human bone morphogenetic protein-2 (rhBMP-2) was incorpo-
rated into the scaffolds and observed to promote the ectopic bone
formation at muscle tissue.74 Microchannelled 3D architectures of
MWCNT–chitosan were further decorated with Pt nanoparticles
and these materials exhibited high conductivity and could be used
as anodes for direct methanol fuel cell.75
Although water-based ceramic slurries and colloidal suspen-
sions have mostly been used, it may be worth mentioning that
a camphene-based freeze-casting route has been studied as an
alternative method to fabricate porous ceramics. Camphene can
be frozen and easily sublimed near room temperature to form a 3D
interconnected porous network. In one study, zirconia, camphene,
and Texaphor 963 as a dispersant were ball-milled at 60 ◦ C for
20 h to form a slurry. This slurry was left to freeze in a mould in
water at 15 ◦ C. Sublimation of camphene at room temperature
and subsequent sintering at 1450 ◦ C produced a ZrO2 foam with
high compressive strength.76 TiH2 powder slurries in camphene
with oligomeric polyester as a dispersant were also prepared by
ball-milling at 60 ◦ C. The warm slurries with TiH2 content of 40,
25 and 10 vol% were poured into an aluminum mould at 42 ◦ C
in sequence at 5 min intervals. During the first 5 min interval, the
40 vol% slurry first poured was frozen to a solid. The slurries with
solid content of 25 and 10 vol% were then in turn poured on
the previously frozen slurry at 5 min intervals. After the removal
of camphene, decomposition of TiH2 under vacuum at 400 ◦ C,
and sintering at 1300 ◦ C, titanium scaffolds with a gradient pore
size and porosity were fabricated.77 It was also possible to pre-
pare aligned porous structures from camphene solutions followed
by directional freezing. For example, polycarbosilane solutions
in molten camphene in polyethylene moulds were directionally
frozen in cool ethanol or liquid nitrogen. The frozen bodies were
placed in a cool atmosphere at around −68 ◦ C to enhance their
green strength. Camphene was then removed by freeze drying
from the frozen samples and the polycarbosilane was crosslinked
via oxidation curing at 200 ◦ C in air. The materials were then py-
rolyzed at 1400 ◦ C to form SiC ceramics with an aligned porous
177
structure.78
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Figure 7. SEM images of honeycomb monolith structures prepared from PLLA/PEG6000 blend (90/10) and 1,4-dioxane solution. (A) Cross-sectioned
surface perpendicular to freezing direction. (B, C) Cross-sectioned area parallel to freezing direction, and (D) the structure of pore wall after leaching with
ethanol.82 (Reprinted with permission from Ref 82, copyright American Chemical Society 2009).
Organic solutions
Organic solutions have been freeze dried mainly for the prepara-
tion of porous hydrophobic biodegradable polymers as scaffolds
for tissue engineering. Porous poly(caprolactone) (PCL) was pre-
pared by freezing a PCL–tetrahydrofuran solution and then freeze
drying at −80 ◦ C. The effects of the concentrations of polymer
on scaffold properties such as morphology, porosity, mechanical
stability, degradability were investigated.79 To produce PCL and
poly(D,L-lactide) with greater control of porosity, polymer solutions
were prepared by dissolving in 1,4-dioxane with the addition of
92 wt% sugar and salt particles.80 Then the freeze drying process
was combined with leaching of the sugar and salt particles.
Initially directional freezing has been used in water-based
systems for the preparation of aligned porous materials.4 – 6 The
scope of this method was significantly enhanced when organic
solutions were used to prepare aligned porous structures. Thus
PCL–dichloroethane solutions were directionally frozen and then
freeze dried to prepare porous PCL with well-defined aligned
microchannels.6 Poly(L-lactic acid) (PLLA) monoliths with aligned
pores could also be obtained by directional freezing of PLA
solutions in 1,4-dioxane.81 In another study by the same group,
PLLA and poly(ethyleneglycol) (PEG) were dissolved in 1,4-dioxane
to prepare a single phase polymer solution. The effects of the
molecular weight of PEG and the PLLA/PEG weight ratio on the
structure of aligned channels and the pores in the channel walls
were investigated.82 Figure 7 shows the aligned channel structure
of the freeze dried PLLA/PEG blend scaffold. After leaching the
PEG with ethanol, smaller pores can be seen in the channel wall
(Fig. 7D). These pore sizes in the channel walls depend on the
molecule weight and concentration of PEG in the solutions.82
Supercritical fluids have been widely used as solvents for
organic synthesis, catalysis, polymer synthesis and extraction.83,84
Among the supercritical fluids, supercritical CO2 (SC CO2 ) has
received particular attention because it is non-toxic, cheap and
178
non-flammable with mild critical points. SC CO2 has been used
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L Qian and H Zhang
as porogen or foaming agent to produce various types of
porous materials.85 Zhang et al. explored the possibility of using
compressed CO2 solution for the directional freezing process.86 A
sugar acetate, 1,2,3,4,6-pentaacetyl-β-D-galactose was dissolved
in liquid CO2 . The resulting solution was then directionally frozen
in liquid nitrogen and the frozen sample left in a fume cupboard
with the valve open to slowly release CO2 . This led to the formation
of a well-defined aligned porous structure. This process involved
no organic solvent and also avoided the step of freeze drying.
Emulsions
An emulsion is a heterogeneous mixture of one immiscible
liquid dispersed into another one in the form of droplets. An
emulsion can be classified as an oil-in-water (O/W) or a water-
in-oil (W/O) emulsion. Emulsions have been used as templates
to prepare porous materials by polymerizing the monomers in
the continuous phase and then removing the solvent from the
droplet phase.87,88 Alternatively, the emulsions can be frozen to
lock in the emulsion-templated structure. Then the solvents in the
continuous phase and the internal droplet phase are removed by
freeze drying to generate porous materials. Whang et al.89 reported
the freeze drying of water/poly(lactic-co-glycolic acid) (PLGA)-
methylene chloride emulsions to form porous biodegradable
PLGA materials. Using the same method, bovine serum albumin
(BSA) and horseradish peroxidase solutions were dispersed into
PLG–methylene chloride solutions to form emulsions. After freeze
drying, porous materials loaded with protein were produced and
used as the matrix for the controlled delivery of protein.90,91
Cameron et al.92 prepared porous PLGA and PCL materials by
freeze drying water-in-oil emulsions using Span 80 as a surfactant.
The adhesion and proliferation of human bladder stromal cells
on the scaffolds were investigated. The surface of porous PLGA
scaffolds was further modified with heparin to deliver growth
factors for soft tissue engineering.93
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Figure 8. Porous SCMC materials prepared by freeze drying the emulsions with different emulsion ratios. From A) to E) the volume percentage of oil
phase is 0, 20%, 40%, 60% and 75%. The white circle indicates one of the emulsion-templated pores.49 (Reprinted with permission from Ref. 49, copyright
Royal Society of Chemistry 2009).

Figure 9. (A) SCMC nanofibres, scale bar 5 um, inset scale bar 600 nm. (B) Hollow titania microtubes, scale bar 10 µm, inset scale bar 1 µm.96 (Reprinted
with permission from Ref. 96, copyright Royal Society of Chemistry 2009).

Combination of emulsion templating and freeze drying to
prepare porous materials has several advantages. For example,
emulsion stability in general is not a problem because the
emulsion structure is locked in by the rapid freezing process.
The percentage of the droplet phase in the emulsion can be varied
over a wide range of 10–95%, thus providing great control on the
pore structure and porosity of the resulting porous materials.
In one study, cyclohexane was emulsified in aqueous SCMC
solutions containing SDS as a surfactant at volume ratios of 0 : 100,
20 : 80, 40 : 60, 60 : 40, and 75 : 25. Freeze drying of the emulsions
produced porous materials with both emulsion-templated and
ice-templated pores (Fig. 8).49 These porous polymeric materials
were further used as templates to prepare porous zirconia with
systematically controlled pore morphology and pore volume.
MICROWIRES AND NANOWIRES
A 3D interconnected fibrous network of PLA could be prepared by a
procedure involving thermally induced gelation, solvent exchange
and freeze drying. Variables such as polymer concentration,
thermal annealing, solvent exchange and freezing temperature
was found that diluted aqueous polymer solutions could be frozen
in liquid nitrogen and then freeze dried to produce polymeric
nanofibres with diameters in the range of 200–600 nm.96 A range
of hydrophilic nanofibres including PVA, SCMC and alginate were
successfully obtained. The polymeric nanofibres could then be
used as templates to prepare hollow crystalline titania microtubes
and Fe2 O3 nanofibres. Figure 9 shows the SEM images of SCMC
nanofibres and hollow TiO2 microtubes.96 Chitosan and chitosan-
based fibres with diameter from 100–700 nm were also produced
by freeze drying 0.1 wt% solutions.97 This method overcame
some disadvantages of the electrospinning methods such as the
requirement for toxic organic solvents or highly concentrated
acetic acid as the solvent. The chitosan nanofibres exhibited good
adsorption capacity for Cu2+ ions in water and could also be used
as scaffolds for the release of Rhodamine B and BSA.
Although the preparation of polymeric nanofibres via a freezing
route was reported only recently, the formation of oxide fibres by
directional freezing had been published decades ago. Mahler and
Bechtold first reported the formation of silica fibres via directional
freezing in 1980.98 A silicic acid solution in water was adjusted to
pH 5.0 using NH4 OH and gelled for 15 min to prepare silica sol.
179

could affect the size of the formed nanofibres.94,95 Very recently, it The silica sol after ageing for 30 min was directionally frozen. After

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 www.soci.org
Figure 10. (A) Gold sub-micron fibres prepared by freezing concentrated gold nanoparticle suspensions.101 (Reprinted with permission from Ref. 101,
copyright Wiley Periodicals 2008.) (B) Composite microwires formed by freezing the silica@PNIPAM colloidal suspensions.103 (Reprinted with permission
from Ref. 103, copyright Wiley Periodicals 2007).
thawing, parallel silica fibres with polygonal cross-sections were
formed. Silicon nitride fibres could be prepared by directionally
freezing water-based silicon nitride slurries and subsequently
freeze drying. Porous silicon nitride with aligned channels
containing fibrous grains protruding from the internal walls were
observed.99 TiO2 hydrogels were synthesized by polymerization
of titanium tetraisopropoxide in an ethanol/water mixture. The
hyrogel was then unidirectionally frozen in liquid nitrogen and
aged at −30 ◦ C for 24 h to strengthen the fibre structures. The
thawed samples were then washed with t-butyl alcohol and freeze
dried to produce TiO2 cryogel fibres.100
The use of aqueous colloidal suspensions for the preparation of
microwires has attracted significant attention recently. Zhang et al.
carried out a study on aqueous suspensions of gold nanoparticles
(average diameter 15 nm), metal oxide nanoparticles (average di-
ameter 20–40 nm) and PS colloids (average diameter 450 nm).101
Gold nanoparticles were aggregated to form microwires with
the diameter around 700 nm (Fig. 10(A)). Microrod structures
were formed with the metal oxide nanoparticles. Surprisingly,
PS colloids also packed together nicely to form microwires. Upon
heating above the glass transition temperature, the PS colloids
fused to form smooth microwires. Freeze-standing nanoparticles-
assembled fibres were also formed by directional freezing of
aqueous PS nanoparticle (80 nm) suspensions by Yan et al.102
Chemical vapour deposition (CVD) was further employed to afford
ductility to these brittle fibres. Stucky and co-workers fabricated
fibres with an ordered brick-and-mortar arrangement of inorganic
phase and organic phase composed of silica (and other inor-
ganic nanoparticles)@poly(N-isopropylacrylamide) (PNIPAM) in a
directional freezing approach.103 Figure 10(B) shows one image
of the fibres made from the silica@PNIPAM core-shell microgels
with 250 nm silica particles and 5% crosslinked PNIPAM. They fur-
ther produced composite fibres from silica@polyacrylonitrile (PAN)
colloidal suspensions.104 Pyrolysis was carried out at 800 ◦ C to car-
bonize the PAN fibres and hollow carbon fibres were formed after
the removal of silica cores with concentrated alkaline solutions.
When another type of inorganic nanoparticles was incorporated
into the starting colloids (e.g. SiO2 @ZrO2 @PAN particles) car-
bon/metal oxide microfibres could be produced.
MICROPARTICLES AND NANOPARTICLES
As mentioned earlier, spray-freezing into liquid (SFL) can
180
produce microparticles19 and has been used to prepare
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c 2010 Society of Chemical Industry
L Qian and H Zhang
microparticles containing poorly water soluble drugs (e.g.
danazol and carbamazepine) either alone or with pharmaceutical
excipients.19,105,106 The dissolution rate of a poorly water soluble
compound can be significantly enhanced by reducing the particle
size or increasing the exposed surface area in water. For high
potency danazol microparticle powders (with drug potency up to
91%), 95% of danazol could be dissolved in only 2 min, compared
with 30% of the danazol being dissolved in 2 min for micronized
bulk danazol.105 Protein and enzyme particles have been pro-
duced by both SFL and spray freeze drying (SFD).107 – 109 In SFD,
a solution was sprayed into a vessel half filled with a cryogenic
liquid. The atomized droplets began to freeze in the cold vapour
and then completely froze in the cryogenic liquid. It was found
that smaller loss of enzyme activity or protein denaturation was
observed for SFL-produced particles owing to the reduced time
of exposure to the air–water interface during atomization.107 – 109
The morphologies of the particles produced by spray freezing
could be engineered in different ways by varying the spray geom-
etry and cooling rate,110 the use of a four-fluid nozzle,111 or using
solid-in-oil-in-oil suspensions as the feed.112 Spray freezing for the
preparation of particles has also been used for food,113 fat,114 and
inorganic particles such as catalysts for oxidation of methane and
CO.115
Hollow biodegradable PLA particles with porous shell walls
have been prepared by both emulsification/freeze drying and
spray/freeze drying. The porous particles were then suspended
in an aqueous solution containing a water soluble drug and
the resulting suspensions were exposed to plasticizing solvents
(dichloromethane or compressed CO2 ). The plasticizing solvent
caused the pores in the shell wall to close and thus encapsulate the
drug in the capsules for future sustained release.116 Alternatively
hollow PS particles with controllable holes in the surface could be
fabricated by a swelling and a freeze drying process. Thus solid PS
colloids were suspended in a mixture of water and toluene and the
toluene could enter and swell the PS colloids. The suspension was
then added dropwise to liquid nitrogen. The frozen sample was
slowly warmed to allow the solvent to evaporate slowly generating
hollow PS colloids with holes on the surface (Fig. 11(A)). Different
functional material such as drugs, proteins, and smaller colloids
could be encapsulated in the hollow PS particles when the holes
were closed by annealing at a temperature slightly above the glass
transition temperature.117 A similar strategy was employed by Yin
et al. After encapsulating the hydrophilic active substances, the
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Figure 11. (A) Hollow PS particles prepared by swelling and freeze drying PS beads in the mixture of water/toluene.117 (Reprinted with permission from
Ref. 117, copyright Nature Publishing Group 2005.) (B&C) Aligned porous PCL particles in aligned porous PVA and Aligned porous PCL microparticles
only.119 (Reprinted with permission from Ref. 119, copyright Wiley Periodicals 2008.) (D) Organic nanoparticles prepared by freeze drying oil-in-water
emulsions.120 (Reprinted with permission from Ref. 120, copyright Nature Publishing Group 2008).

pores on the surface of the hollow PS particles were closed by
exposing to a second swelling solvent.118
An emulsion may also be directly frozen and processed to
prepare porous microparticles. For example, a PCL–xylene solution
emulsified in a PVA–sodium dodecylsulfate (SDS) solution to
form an oil-in-water (O/W) emulsion was directionally frozen
and freeze dried to produce aligned porous microparticles
embedded in an aligned porous matrix (Fig. 11(B)).119 The aligned
porous microparticles could be collected easily by dissolving the
composite material in water and then filtering (Fig. 11(C)). The
porous PCL particles were further tested as scaffolds to support
the growth of mouse embryonic stem cells. There was no evidence
of toxicity over a total culture period of 7 days.119
In other studies, freeze drying of emulsions was investigated to
produce organic nanoparticles. An organic dye Oil Red (OR) was
dissolved in the oil droplet phase of a W/O emulsion with a PVA
solution as an aqueous continuous phase. After freeze drying the
emulsion, OR nanoparticles within the highly porous PVA were
obtained. The porous material could be easily dissolved in water
to form a stable aqueous nanosuspension. The average size of
the OR nanoparticles was around 90 nm (Fig. 11(D)).120 A poorly
water soluble antibacterial agent, triclosan, was dissolved in the
droplets of an O/W emulsion. After freeze drying the emulsion and
dissolving the prepared nanocomposite in water, the resulting
aqueous triclosan nanoparticle dispersion displayed significantly
enhanced biocidal activity compared with the triclosan solution
in water/ethanol.120 Using emulsions could be a general route to
prepare aqueous nanoparticle dispersions of poorly water soluble
organic compounds.121 A high percentage of pharmaceuticals
are poorly soluble in water which limits their bioavailability and
biosorption and this technique may therefore be employed to
significantly enhance their water solubility.105,106 In addition to
releasing the organic nanoparticles in water by instant dissolution,
the organic nanoparticles could be formed in a temperature-
remain inside PNIPAM at room temperature and then could be
released into water in a burst by raising the temperature above
32 ◦ C (the low critical solution temperature for PNIPAM). Several
cycles of temperature-sensitive release were demonstrated.122
CONCLUSIONS
The processes of freezing and freeze drying have been widely used
in materials synthesis and applications. When a frozen solution
(or suspension or emulsion) is freeze dried, the frozen solvent is
sublimed to produce dry or solvent-free structures. The low surface
tension involved during the freeze drying process can maintain
the pore structure (particularly mesopores and micropores) which
would otherwise collapse due to the high capillary force or surface
tension during a normal drying process. For this reason, freeze
drying has been employed frequently in the preparation of porous
materials. For example, it was found recently that freeze drying
could significantly increase permanent porosity and H2 uptake of
metal–organic frameworks.123
The distinct advantage of the freeze drying method may be the
use in water-based systems which are essential for biological and
environmental applications. Freeze drying processes may avoid
the use of toxic organic solvents and the low temperature of the
process helps maintain the activity of biomacromolecules (such as
proteins, enzymes) and pharmaceuticals. Freeze drying has thus
been exploited in various novel processes. For example, freeze
drying of aqueous solutions was used to produce deep eutectic
solvents (DES) and the incorporation of liposome with preservation
of their self-assembled structure in DES demonstrated.124 Bacteria
could also be incorporated in DES in its pure state with the
preservation of bacteria integrity and viability.125 By freeze drying
aqueous solutions of cationized ferritin and an anionic polymeric
surfactant, Mann et al. produced ionic nanostructures that could
then be melted at 50 ◦ C to obtain solvent-free protein liquids.126
181

responsive porous crosslinked PNIPAM.122 The nanoparticles They further demonstrated the preparation of room-temperature

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 www.soci.org
solvent-free myoglobin liquids without significant loss of activity
and the reversible dioxygen binding properties in the liquid
myoglobin.127
This review has focused on the use of freezing and freeze
drying for the preparation of porous and micro-/nano-structured
materials. Solutions, emulsions, and colloidal suspensions can
be processed to obtain a wide range of porous structures
and particulate materials. Morphologies of these materials
are determined during the freezing process with the freeze
temperature, nature and concentration of solute and solvent, and
freeze direction significantly affecting the morphology. Among
methods of controlled freezing, directional freezing has been
developed to prepare aligned structures and spray freezing
can be used to form porous microparticles. The combination
of emulsion templating and freeze drying has offered great
control of pore morphology and pore volume and also led to
the formation of organic and pharmaceutical nanoparticles within
the porous materials. The materials demonstrated in this review
cover different morphologies (porous structures, microwires and
nanowires, microparticles and nanoparticles) and various types of
materials (polymers, proteins, pharmaceuticals, metals, silica, and
metal oxides).
Owing to the advantages and versatility of the freeze drying
method, it is believe that this route will be continuously
investigated for novel processes and applications. With regard to
the porous materials and nanostructured nanomaterials, we feel
that research will advance in the following directions: (i) materials
with novel structures, hierarchical porosity and mechanical
strength; (ii) purposely-engineered materials with defect-free
uniform structures on a relatively large scale; (iii) biocompatible
and biodegradable structures and particles with controlled
morphology, surface functional groups and tunable mechanical
stability for three-dimensional tissue engineering, directing cell
growth and controlled delivery; (iv) functional materials (including
polymers, inorganics and semiconductors) for catalysis, energy
storage and fuel cells.
ACKNOWLEDGEMENT
This work was supported by the EPSRC (EP/F016883/1).
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