Review

Thermoresponsive polymers as
gene and drug delivery vectors:
architecture and mechanism of
1. Introduction
2. Polymers with a lower critical
action
solution temperature
Maria Teresa Calejo, Sverre Arne Sande & Bo Nystr€om†
3. Temperature-responsive †
University of Oslo, Department of Chemistry, Oslo, Norway
hydrogels
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4. Temperature-responsive Introduction: This topic is important as it allows for improved specificity in

polymers as gene delivery drug delivery, providing possibilities for reduced side effects, and thereby
vectors improved pharmacotherapy. As a wealth of different polymers and mecha-
5. Conclusion nisms of action has been suggested, a systematic overview of the field is of
current importance.
6. Expert opinion
Areas covered: This article presents an overview over a selection of thermor-
esponsive polymers suitable as excipients in systems for gene and drug deliv-
ery with particular emphasis on the influence of polymer structure,
composition, molecular weight (MW) and architecture on the responsive
mechanisms. Due to the immense number of reports on these increasingly
popular materials, focus has been restricted to the use of micelle-forming pol-
ymers with a lower critical solution temperature, temperature-responsive
For personal use only.

hydrogels for drug delivery applications and temperature-sensitive polymers

as non-viral vectors for polynucleotide delivery. Specific examples covered
are poly-(N-isopropylacrylamide) (PNIPAAM), Pluronics and their derivatives.
It is concluded that the studies constitute an excellent platform for develop-
ment of thermoresponsive systems with improved gene and drug
delivery properties.
Expert opinion: A thorough knowledge of factors important for loading
efficiency and drug release is necessary to be able to develop optimal
nano-carriers for the future. Other issues that are not fully understood is
how small the carriers need to be, and which manufacturing procedures
should be used.

Keywords: drug delivery, gene transfer, hydrophilic--hydrophobic balance, polymer--drug

interaction, temperature-responsive copolymers

Expert Opin. Drug Deliv. (2013) 10(12):1669-1686

1. Introduction

1.1 Polymer therapeutics

Since the start of drug delivery formulation, large polymeric molecules have been
vital for obtaining the desired properties of the system. From the initial use as inert
diluents or binders in capsules and tablets providing improved technical properties,
the versatility of polymeric substances has led to the possibility of introducing
functionality to the drug delivery systems, allowing accurate control of the time,
place and amount of drug release. Examples of such systems range from the sus-
tained release tablets providing the possibility for once-a-day dosing, via site-specific
drug delivery and drug targeting, to stimuli-responsive systems capable of
modulating the drug release rate according to one or more external factors.
The molecules of a polymer consist of a large number of connected smaller units,
the monomer units, joined by covalent bonds. As such, they are generally flexible

10.1517/17425247.2013.846906 © 2013 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 1669
All rights reserved: reproduction in whole or in part not permitted
 M. T. Calejo et al.
Article highlights.
. Temperature-responsive copolymers are versatile and
attractive for drug delivery and gene transfer.
. The loading efficiency and drug release are governed
by factors such as polymer architecture, composition of
hydrophobic and hydrophilic sequences in the
copolymer, sign and charge density, and MW.
. The circulation time of the carriers in the blood stream
can be significantly extended if they are properly
stabilized by PEG.
. To achieve a controlled drug release, the polymer--drug
interaction at different temperatures is crucial and can
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 12/01/13
be regulated through the structure and composition of
the copolymer carrier.
. There are some positively charged temperature-sensitive
copolymers, based on poly(N-isopropyl acrylamide)
(PNIPAAM), that are promising for gene delivery
applications.
. There are some findings suggesting that it is possible to
increase the biodegradability and decrease the toxicity
of the carriers by changing the copolymer composition.
This box summarises key points contained in the article.
For personal use only.
objects, which resemble coils of chains. At some conditions,
certain types of polymer may adopt a more rigid and rod-
like shape; other sorts of polymer may roll up into a compact
structure resembling a sphere. In general, polymers find broad
uses in gene and drug delivery applications. Some obvious rea-
sons for this are their surface activity that makes them efficient
stabilizers of colloidal drug systems, their capacity to form
hydrogels that are useful for many formulations, as well as
their ability to self-assembe into complex structures that are
similar to those formed by classical low-molecular-weight sur-
factants. In drug- and gene delivery, several different types of
polymers have attracted a great deal of interest, but amphi-
philic block copolymers, consisting of blocks of two or more
polymer types of different hydrophilicity, have been found
useful for similar reasons as low-molecular-weight surfactants.
A very old example of a functional polymer employed in drug
delivery and extensively commercialized is the use of polyme-
thacrylic acid for coating of tablets which thereby provide
resistance towards gastric fluids. A more recent example is
the coating of liposomes with polyethylene glycol (PEG) pro-
viding stealth properties to the liposomes, thereby allowing
increased circulation time by avoiding the cells of the reticu-
loendothelial system (RES). Even for nanoparticles that are
not polymer-based such as gold nanoparticles, polymeric sur-
face functionalization plays an important role. For instance,
polymers may act as linkers between the particle surface and
targeting molecules intended to direct the particle to specific
organs or cells. Targeting cancer cells is often achieved, for
example, using monoclonal antibodies against molecules
enriched on the surface of cancer cells such as folate or trans-
ferrin receptors [1].
1670 Expert Opin. Drug Deliv. (2013) 10(12)
The primary requirement for pharmaceutical excipients in
general is lack of toxic or other pharmacological activity,
biocompatibility and, especially important for polymers,
biodegradability. The advantages of polymers in drug delivery
primarily stems from their size, allowing complete encapsula-
tion of small therapeutic molecules, either to protect the
active substance from the body (improved stability) or to pro-
tect the body from the drug (reduced side effects). But also for
larger therapeutic substances such as proteins or nucleotides,
polymers play an important role. In these cases, several poly-
meric molecules are required and the ability of the polymers
to self-assemble becomes important. Typically, amphiphilic
polymers self-assemble through hydrophobic associations. In
addition, ionic interactions between oppositely charged
polymers and chemical (covalent) cross-linking of polymers
have been suggested as drug delivery systems. Examples of
self-assembling systems are micelles, polymersomes [2] and
core-shell nanoparticles [3].
Besides size, versatility in structure is important for the
popularity of these systems. The copolymers may be ran-
dom-, block- or grafted on a backbone, allowing a multitude
of polymer--polymer or polymer--drug interactions to occur,
in addition to a fine-tuning of drug delivery properties.
1.2 Stimuli-responsive polymers
In recent years, stimuli-responsive polymers (also called
‘intelligent’ or ‘smart’ polymers) have attracted great attention
for drug delivery applications. These polymers respond to
small changes in environmental conditions, such as tempera-
ture, pH, redox potential, light, ionic strength, electric or
magnetic fields, or the presence of enzymes or specific
ligands [4]. In addition, some polymer systems can combine
more than one responsive mechanism, for example by
responding to both pH and temperature changes [5,6]. The
possibility to change the physicochemical properties of the
system as a response to a specific stimulus makes the smart
polymers appealing in a number of applications, for instance
to promote the adhesion/detachment to proteins and cells in
cell culture engineering and bioseparation [7,8], forming scaf-
folds in tissue engineering applications [9,10] and as responsive
delivery systems for drugs [11-13] and nucleic acids [14-16].
In drug delivery, polymers that respond to pH variations or
temperature changes are particularly interesting materials,
since their ability to undergo reversible phase transition
or conformational changes depending on the external
environment can be used to promote the drug loading, and
to modulate the rate and site of drug release [4].
Polymers that respond to temperature changes are among
the most investigated in modern drug delivery applications
and they are characterized by changing their conformation, sol-
ubility and hydrophilic/hydrophobic balance as a response to
changes in the temperature of the external environment [4,17].
In this article, the use of temperature-responsive polymers
in drug and gene delivery is discussed. Particular emphasis
will be given to the influence of polymer structure,
 Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action
composition, molecular weight (MW) and architecture on the
mechanism of action of the system. Specific examples of pol-
ymers that undergo reversible volume phase transitions and
sol-gel transitions will be presented, such as poly(N-isopropy-
lacrylamide) (PNIPAAM) and Pluronics, and their copoly-
mers. A short summary of illustrative examples is provided
in Table 1. Due to the immense number of reports on these
increasingly popular materials, a comprehensive review of
the literature would exceed the limits of this article. Focus
will therefore be restricted to the use of thermoresponsive pol-
ymers in micelles and hydrogels for drug delivery applications,
and as non-viral vectors for polynucleotide delivery.
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For clarification to the reader, some of the most common
polymer architectures mentioned in the text are depicted
in Figure 1.
2.Polymers with a lower critical solution
temperature
Some temperature-responsive polymers are characterized by
having a lower critical solution temperature (LCST). The
LCST can be defined as the critical temperature above which
the polymer solution undergoes a macroscopic phase separa-
tion [18]. Below this temperature, the polymer chains are
For personal use only.
water-soluble and usually form hydrogen bonds with the water
molecules. As the temperature is raised above the LCST, the
hydrogen bonds are broken and the hydrophobic associations
dominate, causing the polymer in dilute solution to undergo
coil-to-globule transition, resulting in macroscopic phase sep-
aration at higher polymer concentrations. An illustration of
the effect of temperature on the phase transitions of
temperature-responsive polymers is shown in Figure 2A. In
this process, the balance between the ratio of hydrophobic
and hydrophilic monomers is of great importance [4,17,19].
2.1 Poly(N-isopropylacrylamide) and its copolymers
One of the most extensively studied thermoresponsive poly-
mers is PNIPAAM. This polymer exhibits a phase transition
at about 32  C in water, depending on its concentration
and MW [20]. This proximity to human body temperature
makes it particularly interesting for pharmaceutical and bio-
medical applications [21]. Copolymerization with other blocks
can result in combined properties, as in the case of the copo-
lymerization of PNIPAAM with poly(acrylic acid) for both
temperature- and pH-responsiveness [22]. The main limitation
of PNIPAAM is its non-biodegradability, but making copoly-
mers with other biodegradable polymers may partially con-
tribute in overcoming this issue [23]. By copolymerization
with other hydrophilic or hydrophobic blocks, one can alter
the LCST of the polymer, or produce distinct micellar struc-
tures for specific applications [24-26]. Further, the cloud point
(or the LCST) can be tuned by incorporating charged blocks
into the copolymer [27,28]. It has been suggested that no toxic-
ity is caused by using low MW PNIPAAM, as the polymer is
easily eliminated by glomerular filtration [29]. This emphasizes
Expert Opin. Drug Deliv. (2013) 10(12)
the importance of polymer MW in the design of new poly-
mers for drug delivery applications.
Owing to the widespread importance of polymeric
micelles as drug delivery systems, the self-assembling prop-
erties of PNIPAAM copolymers into micelles will be espe-
cially highlighted. Polymeric micelles can form nano-sized
structures (generally 10 -- 200 nm) and the combination
of a hydrophobic core and a hydrophilic corona provides
them with unique properties. The hydrophobic core can
accommodate hydrophobic drugs, which may be loaded
during micelle formation and the hydrophilic corona pro-
vides the drug carrier with shielding properties, contribut-
ing to reduced recognition by RES and increased
circulation time. Accumulation in diseased or inflamed tis-
sues such as tumors can take place through the enhanced
permeability and retention (EPR) effect, making the
micelles especially advantageous in cancer [19,23,30-32]. Addi-
tional advantages are the ease of manipulation and the
possibility to functionalize the micelles, for example, for
targeting effects [19,30-33].
Below the LCST, copolymers of PNIPAAM with hydro-
phobic segments can form core-shell micelles, where the
core is formed by the hydrophobic domains and the shell is
composed of the hydrated PNIPAAM. As the temperature is
increased above the LCST, the hydrophobic groups of PNI-
PAAM associate, causing destabilization and aggregation of
the micelles and triggering a fast drug release (Figure 2B) [23].
This reversible and thermo-sensitive response has been
shown for adriamycin-loaded PNIPAAM-b-poly(butylmetha-
crylate) [34], docetaxel-loaded (PNIPAAM-co-acrylamide)-b-
poly(DL-lactide)) [35] and for pyrene-loaded PNIPAAM-co-
polystyrene micelles [3], demonstrating the potential of these
polymeric systems for the delivery of hydrophobic drugs,
particularly in combination with localized hyperthermia. In
antitumor drug delivery, this strategy can be particularly
advantageous. Docetaxel-loaded micelles, for instance, have
shown to be much less cytotoxic than the free drug, contribut-
ing to a decreased systemic toxicity in vivo. Further, by
local heating of the tumor site, the cytotoxicity of docetaxel-
loaded micelles increased to a level similar to that of the free
drug [35].
Liu et al. [36] synthesized PNIPAAM-co-N-N-dimethylacry-
lamide-b-poly(D,L-lactide-co-glycolide) ((PNIPAAM-co-
DMAAm)-b-PLGA)) to produce micelles containing doxoru-
bicin (DOX) for anticancer drug delivery. The hydrophilic
DMAAm was introduced to adjust the LCST of the copoly-
mer, whereas the hydrophobic PLGA was the core-forming
segment of the core-shell micelles. A balanced hydrophilicity
was shown to be important in order to maximize the drug
loading efficiency, and a faster drug release rate was observed
at temperatures above the LCST of the copolymer, because of
the collapse of the outer PNIPAAM shell. However, the pres-
ence of longer PLGA blocks seemed to slow down the process,
due to a higher polymer aggregation number per micelle,
creating stronger PLGA--DOX interactions [36].
1671
 M. T. Calejo et al.

[69,70]
Refs.
Poly(benzyl ether)-PNIPAAM dendritic-linear diblock

[122]

[128]
[34]
[35]

[80]
copolymers based on a second- or third-generation poly
(benzyl ether) dendrimer ([G-2] or [G-3]) were prepared [37].

Hyperthermia in early stage causing DNA compaction

Structural changes due to collapse of PNIPAAM shell

G-3-PNIPAAM self-assembled in aqueous solution into

Advantages of thermoresponsive properties

micelles consisting of a dendritic core and a PNIPAAM

above the transition temperature, allowing drug

Active targeting by thermal precipitation of the

conjugates. Drug release facilitated by low pH
shell. The results suggested that guest hydrophobic mole-

and high temperature (due to dissociation of

Accumulation and aggregation of polyplexes

in tumor tissues by local hyperthermia and

Nanocapsule swelling at low temperatures

Hypothermia in a later stage causing DNA
cules could be taken up by the hydrophobic core, being

release and facilitating endosomal escape

located in the void space between the dendrons. Impor-
Drug loading by temperature increase

hyperthermia/hypothermia protocol:
tantly, the authors suggest that, at low temperatures, the
porous dendritic core allows the small hydrophobic mole-

Increased gene expression by

cules to quickly diffuse in and out of the core, whereas

enabling endosomal escape

increased gene expression
the collapse of PNIPAAM at higher temperatures causes
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the pores to contract, thus limiting the diffusion of guest

release upon heating

molecules. As compared to the linear block copolymers,

the dendritic blocks can form highly ordered, non-
the conjugate)

entangled cores, and can have a high loading capacity due

Table 1. Examples of thermoresponsive systems and their advantages in drug and gene delivery applications.

to the presence of void spaces [37]. Only a small number

of other ‘smart’ dendrimers have been reported, for instance
based on a poly(propylenimine) core decorated with PNI-
PAAM arms [38] and on a dendritic poly(ether-amide) core
Bio-active molecule

forming a star-shaped polymer, containing poly(-caprolac-

tone) (PCL)-PNIPAAM arms (PCL acting as the inner
Small interfering
Geldanamycin

hydrophobic block) [39]. Despite the potential of stimuli-

(conjugated)
Adriamycin

responsive dendrimers in drug delivery, reports on in vivo

Docetaxel

Paclitaxel
For personal use only.

studies concerning these systems are still quite scarce, and

DNA

DNA

RNA

their synthesis is considered expensive [40].

ABA-type block copolymers (consisting of a central block B
and two terminal A blocks, Figure 1) based on PNIPAAM
Nano-constructs

Nanocapsules

with a short central hydrophobic segment have also been

Carrier

Polyplexes

Polyplexes

reported to form core-shell micelles in aqueous medium.

Micelles
Micelles

Micelles

The triblock structure has been suggested to result in higher

drug loading capacities and higher thermo-sensitivity as com-
pared to the diblock counterparts [29]. Studies on PNIPAAM-
AB-type block copolymer
AB-type block copolymer

AB-type block copolymer

AB-type block copolymer

PCL-PNIPAAM block copolymers showed that as the length

of PNIPAAM is increased, the critical micelle concentration
Statistical copolymer
Architecture

(CMC) also increases due to the higher hydrophilicity of the

cross-linked to PEI
Block copolymer
Graft copolymer

(PEO--PPO--PEO)

polymer, and the micelles become larger due to increased

grafted to PEI

hydration of the shell at temperatures below the LCST.

A lower capacity for incorporation of hydrophobic substances
is, however, expected in this case. At higher temperatures,
micelle aggregation takes place because of the collapse of
PNIPAAM [41]. In the context of drug delivery, this study
(PNIPAAM-co-acrylamide)-b-poly(dl-lactide))

PEI-g-poly(NIPAAM-stat-vinylpyrrolidinone)
Poly(N-(2-(hydroxypropyl) methacrylamide

demonstrates the influence PNIPAAM length on the hydro-

philic--hydrophobic balance, affecting the size and loading
behavior of the micelles. Moreover, the importance of the
MW was demonstrated. In a previous study, the presence of
PNIPAAM-b-butylmethacrylate

a long PCL segment (Mn ~ 42,000 g/mol) was shown to

render the triblock copolymer insoluble in water [41,42].
The triblock PCL--PNIPAAM--PCL copolymer was shown
Poly(Asp)/(Glu)-b-ELP

to self-assemble into core-shell micelles and was loaded with

dilactate-b-PEG

PEI-g-PNIPAAM

the anti-inflammatory drug prednisone acetate [29]. The

hydrophobic drug was released very fast as the temperature
Pluronic/PEI

was increased to 37 C, as a result of the abrupt collapse of

Polymer

the shell and deformation of the micelles. Even though a lon-

ger length of PNIPAAM was shown to cause an increase in

1672 Expert Opin. Drug Deliv. (2013) 10(12)

 Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action
AB-type block copolymer Graft copolymer
ABA-type triblock copolymer
Star copolymer
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Y-shaped copolymer
Dendritic-linear diblock copolymer
Figure 1. Some of the most common polymer architectures
employed in thermoresponsive systems for drug delivery
applications.
the CMC, the influence of block length on the drug loading
For personal use only.
and release was not clearly demonstrated in this study [29].
Only a few authors have studied the formation of core-shell
structures by graft copolymers and investigated their use in
drug delivery. Lo et al. [43] synthesized temperature- and
pH- responsive graft copolymers based on poly(D-L-lactide)-
graft-PNIPAAM-co-methacrylic acid). The length of the
poly(D-L-lactide) (PLA) segments had a pronounced effect
on polymer interactions and on the size of the structures.
The nanoparticles showed to collapse and aggregate in acidic
environment at 37.5 C, due to the thermoresponsive behav-
ior of the polymer and deionization of the carboxylic groups
of methacrylic acid at low pH. In vitro studies on cancer cells
suggested that the nanoparticles experienced deformation in
contact with the low pH environment of the endosomal fluid.
In consistency, drug release studies on nanoparticles loaded
with the anticancer drug 5-fluorouracil showed that drug
release occurred faster at low pH, as a consequence of the col-
lapse of the structure. This leakage behavior was suggested to
be a characteristic of the graft structure of the copolymer. The
system was shown promising as an intra-cellular delivery
vehicle for both hydrophobic and hydrophilic drugs in cancer
therapy [43].
Studies on star [44-46] and Y-shaped [47] copolymers contain-
ing hydrophobic moieties and PNIPAAM similarly showed
self-assembly in aqueous medium, and that drug release
from the micelles occurs faster when these are incubated at
temperatures > LCST.
In most of the above-described studies, core-shell micelles
were formed as long as both hydrophobic and hydrophilic
(in this case PNIPAAM) segments are present. The architec-
ture of the copolymers per se (linear diblock, triblock and
Expert Opin. Drug Deliv. (2013) 10(12)
graft, dendrimeric, star- and Y-shaped) does not appear to
be as vital for the self-association of the copolymers. Con-
versely, factors such as the length of the segments, the hydro-
philic--hydrophobic balance and the charge density have been
shown to influence the formed structures. For instance, a very
long hydrophilic PNIPAAM may compromise micelle forma-
tion and non-structured aggregates might be formed
instead [43,48]. Core-shell micelles of small size can be obtained
if the hydrophobic and hydrophilic moieties are bal-
anced [43,48]. These micelles can demonstrate increased stabil-
ity, for example, small change in particle size and aggregation
number upon heating, when compared to the aggregated
structures [48]. Besides, the LCST of the copolymer and the
size of the micelles can be modified as the hydrophobic moi-
ety (or the length of the segments) is changed [23,39]. These
properties can be used to design intelligent drug delivery sys-
tems with control of drug release induced by temperature
changes. In this context, if the duration of release and loading
efficiency are important, the compactness of the core and the
drug--polymer interactions should be especially considered.
When PNIPAAM is copolymerized with hydrophilic seg-
ments, an overall hydrophilic copolymer with an extended
conformation is obtained at temperatures < LCST. If the tem-
perature is raised above this threshold, micellization will take
place as a result of the hydrophobic associations of PNI-
PAAM, which will constitute the compact core of the
micelles. In this scenario, the hydrophilic segments constitute
the outer corona (Figure 2C). Another consequence of the
presence of a permanently hydrophilic segment is the com-
monly observed increase of the LCST [19,23].
In drug delivery technology, the reversible behavior of ther-
moresponsive copolymers formed by dual hydrophilic seg-
ments can be used to facilitate the loading process of a
hydrophobic drug by heating the solution to a temperature
higher than the LCST, and to promote drug release by
employing a cooling procedure [23,49]. In cancer therapy, for
instance, a burst of the loaded drug can take place at the
tumor site by applying a local hypothermia procedure [49].
However, the need for two temperature protocols can imply
additional difficulties, which may explain why only a small
number of authors report the development of PNIPAAM-
based hydrophilic copolymers.
One of the most well-studied hydrophilic polymers that
have been copolymerized with PNIPAAM is PEG or poly
(ethylene oxide) (PEO). In drug delivery, PEG accounts for
significant advantages, since it is considered biocompatible
and known to interact poorly with plasma proteins and cell
membranes, thereby contributing to increased circulation
time of the carrier system [50,51]. The presence of a hydrophilic
cover on particles and self-assembled structures also prevents
the aggregation of the particles with one another, improving
the stability of the formulation [50,52].
Studies on PNIPAAM-b-PEO copolymers have confirmed
the formation of a spherical core-shell structure, as the tem-
perature was increased above the LCST of PNIPAAM [26,52].
1673
 M. T. Calejo et al.

T < LCST T > LCST

A.

Hydrophobic segment

B. PNIPAAM
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Drug

Hydrophilic segment

C. PNIPAAM

Drug
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Figure 2. Effect of the lower critical solution temperature (LCST) on the phase separation behavior of PNIPAAM and on the
micellization of PNIPAAM copolymers. A. PNIPAAM undergoes a ‘coil-to-globule’ transition above the LCST, as a result of
hydrophobic associations. B. PNIPAAM copolymers containing a permanently hydrophobic segment form core-shell micelles in
aqueous solution at low temperatures. The micelles contain a hydrophobic core and a hydrated PNIPAAM shell. As the
temperature is increased above the LCST, the PNIPAAM shell collapses, causing the destabilization of the micelles and
triggering drug release. C. PNIPAAM copolymers containing hydrophilic segments are water soluble < LCST of PNIPAAM but
the segments self-assemble into micelles at higher temperatures as a result of the self-association of the thermoresponsive
moieties. Core-shell micelles, based on a PNIPAAM core and on a hydrophilic shell are thereby formed. In this case, drugs can
be loaded (or released) if a hyperthermia (or hypothermia) procedure is applied.

Longer PEO blocks contributed to increase the overall hydro-
philicity of the copolymer, which shifted the cloud point to
higher values. Further, when the length of PEO is kept con-
stant, the size and MW of the aggregates seem to increase
with increasing PNIPAAM length (and increasing N-isopro-
pylacrylamide/ethylene oxide ratio), in agreement with a
higher tendency towards aggregation at temperatures
> LCST. Higher polymer concentrations were similarly found
to contribute to increase the size of the aggregates, the number
of polymer chains per aggregate and, consequently, the
density of the structures [52].
Notably, the results of these studies on the block copolymers
suggested that their aggregation behavior was different from
that taking place with similar copolymers with a graft architec-
ture, where the distribution of the PEO grafts along the PNI-
PAAM chains (as well as the degree of grafting) can strongly
affect the thermoresponsive behavior of the copolymers [52,53].
Qiu et al. showed that a lower number of PEO grafts leads
to the formation of larger particles [54]. This is because
copolymers with a low number of PEO grafts (and accord-
ingly lower MW) undergo inter-chain associations even
before the intra-chain coil-to-globule transition takes place.
Smaller particles can additionally be prepared if low copoly-
mer concentrations and fast heating rates are used, since these
conditions can minimize inter-chain aggregation (with pre-
dominance of intra-chain associations) [54]. Due to the recog-
nized effects of particle size on the in vivo distribution,
biological fate and stability of the nano-carriers, as well as
on the toxicity, targeting ability, drug loading and drug
release [55], the control of grafting and carrier preparation con-
ditions is therefore of the utmost importance in drug
delivery applications.
Further, in recent years, multi-responsive or functionalized
systems based on PNIPAAM-PEG copolymers have been
investigated. Studies on a PEG-b-poly(4-vinylpyridine)-b-
PNIPAAM triblock copolymer with both pH- and
temperature-responsive properties showed that three different
micelle structures could be formed by the same copolymer,

1674 Expert Opin. Drug Deliv. (2013) 10(12)

 Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action
depending on the low or high solubility of poly(4-vinylpyri-
dine) at high and low pH, respectively, and on the
temperature-dependent aggregation of PNIPAAM [56].
Functionalized PNIPAAM-g-PEG copolymers containing
an aldehyde group at the PEG chain end have been prepared
so as to allow the conjugation with bio-components such as
antibodies, enzymes, inhibitors and oligonucleotides [50].
Another elegant copolymer was synthesized by You and Oup-
ický [57], consisting of an Y-shaped copolymer of PNIPAAM
and PEO, functionalized to allow conjugation to a ligand.
In this work, biotin was the selected molecule for conjugation
to the thiol group, while a reactive carboxylic acid group was
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introduced so as to enable further modifications or facilitate
the attachment of the copolymers to the surfaces of particles
or various substrates. The stimulus-controlled exposure of
the ligand was demonstrated and related to the micellization
process [57].
Other PNIPAAM copolymers containing hydrophilic
blocks have shown a temperature-dependent gelling capacity,
based on the formation of physical cross-links or on micellar
aggregation mechanisms [58]. Some representative examples
will be presented and discussed in Section 3.
For personal use only.
2.2Poly(ethylene oxide)/poly(propylene oxide)
copolymers
Pluronics or Poloxamers are a family of ABA-type triblock
copolymers with the composition PEO--PPO--PEO and are
commercially available in a wide spectrum of MWs and
poly(ethylene oxide)/poly(propylene oxide) or PEO/PPO
ratios; these variations imply differences in the cloud point
and CMC values, depending on the hydrophilic--lipophilic
balance of the copolymer [59]. Pluronics show good cell
compatibility and low irritation effects following topical or
parenteral administration [60].
The self-assembly behavior of PEO-PPO-PEO triblock
copolymer is strongly correlated to polymer concentration.
At sufficiently high concentrations (i.e., at the CMC), the
unimers arrange into core-shell micelles formed by a hydro-
phobic PPO core and a hydrophilic PEO corona. Long
PPO blocks lead to a decrease in the CMC, due to the higher
hydrophobicity of the copolymer, while the opposite is
observed at longer PEO lengths. The self-association of Plur-
onics is also promoted by higher temperatures, which trigger
the dehydration of PPO, leading to reduced solubility of
this segment [59]. The physicochemical characteristics and
pharmaceutical/bio-medical applications of Pluronics (e.g.,
in cancer therapy and drug solubilization) have been
addressed in other review articles [59,61,62]. In drug solubiliza-
tion applications, the hydrophobic drugs can be solubilized
by being located in the hydrophobic core of the micelles. In
this context, Pluronics with longer PPO blocks (more hydro-
phobic) and higher MW are usually stronger solubilizing
agents [60] of hydrophobic drugs.
Expert Opin. Drug Deliv. (2013) 10(12)
Despite generally being regarded as biocompatible,
PEO--PPO--PEO block copolymers have some important
drawbacks that may limit their applicability, such as their
weak mechanical strength, rapid dissolution and non-bio-
degradability. In vivo, the micelles can quickly dissociate
because of dilution, resulting in unimers that are typically
eliminated by renal filtration [59,62]. Specific synthetic strategies
have therefore been discussed in order to increase the stability
of formulations intended for drug delivery applications [63,64].
Pluronics can additionally form gels in aqueous solutions.
Significantly higher concentrations are however needed in order
to achieve gelation (> 15 wt%), as compared to the micellization
concentrations (< 1 wt%) [60]. The gel-forming properties of
Pluronics will be addressed in the following section.
While a number of Pluronics are Food and Drug Adminis-
tration (FDA) approved and well established in pharmaceuti-
cal applications, their X-shaped counterparts are still poorly
investigated. PEO and PPO copolymers with this architecture
are called poloxamines or Tetronics and are characterized by
having four branched arms, each of them consisting of
PEO--PPO blocks connected to a central ethyleneimine
group [60,65]. The presence of a central group containing two
tertiary amine groups renders the copolymer sensitive to
both temperature and pH changes, and its presence is
additionally suggested to increase the thermal stability of the
copolymer and to offer the possibility for chemical modifica-
tions and cross-linking [60]. The self-associating behavior of
poloxamines is strongly dependent on the MW, ethylene
oxide/propylene oxide (EO/PO) ratio and on the hydrophi-
lic--lipophilic balance of the copolymer. Likewise, factors
such as the pH and ionic strength of the medium are known
to affect the protonation of the amine groups and thus to
influence the hydrophobicity of the copolymer and the self-
assembly process [66]. Recently, it was demonstrated that
hydrophilic, high MW poloxamines were less toxic, whereas
no clear dependence was demonstrated with respect to the
CMC of the copolymer [67]. Gonzalez-Lopez et al. revealed
that high MW poloxamines containing a higher PO/EO ratio
formed larger micelles with more hydrophobic cores, which
thereby are more adequate to host hydrophobic molecules [66].
Long poloxamine molecules containing large PPO blocks may
contribute to enhanced micelle stability upon dilution [66].
Improved solubility of hydrophobic drugs by incorporating
the drug molecules into poloxamine micelles has been demon-
strated using quercetin, a hypollipidemic drug, and both
hydrophobic and electrostatic forces were found to play a
role for the interactions with the drug. Polymer concentra-
tion, salt concentration and pH had a strong effect on the
solubilizing ability of the micelles and the drug release [65].
2.3 Other synthetic polymers with a LCST
Other thermoresponsive polymers have been investigated
as micellar drug carriers. Soga et al. synthesized poly(N-
(2-hydroxypropyl) methacrylamide mono/di lactate
(pHPMAm-ML/DL), a new class of biodegradable polymers
1675
 M. T. Calejo et al.
with tuneable thermosensitivity [68]. Block copolymers of
pHPMAm-DL with PEG are water-soluble at temperatures
< LCST, but they form micelles above this temperature.
The micelles were formed by a pHPMAm-DL core with a
PEG shell, since the first was shown to become hydrophobic
at higher temperatures [69]. In subsequent work, the authors
took advantage of this thermoresponsive behavior to load
the anticancer drug paclitaxel (PTX) into the micelles by
simply increasing the temperature above the critical micelle
temperature of the polymer [70]. The micelles were shown to
efficiently solubilize the hydrophobic drug, which remained
stably incorporated in the micelles at 37 C and pH 7.4, and
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to be stable in the presence of serum proteins. In vitro studies
on a cancer cell line demonstrated that the micelles were suc-
cessfully internalized. Importantly, they exhibited comparable
cytotoxicity to the Taxol standard formulation, while the
drug vehicle in the absence of drug was less toxic than the
commercial counterpart, suggesting that the thermorespon-
sive micelles can be promising delivery systems of PTX [70].
However, the effects of copolymer architecture on the self-
assembling properties and drug delivery potential of the
system have not been evaluated in detail so far.
2.4 Elastin-like polypeptides
For personal use only.
In recent years, the advent of recombinant DNA technologies
has boosted the design and development of recombinant
amino acid based polymers, characterized by high monodis-
persity, well-defined monomer sequence, and allowing
incorporation of multiple functionalities in a controlled,
sequence-specific manner [71].
The biocompatibility and biodegradability and biomateri-
als of ELPs make them particularly appealing biomaterials
for various bio-medical applications [72]. Characteristically,
elastin-like polypeptides (ELPs) based on the amino acid
sequence Val--Pro--Gly--Xaa--Gly undergo spontaneous revers-
ible phase transition in response to temperature alterations, in
a way that resembles the behavior at LCST of PNIPAAM. In
this case, however, the process known as inverse temperature
phase transition (ITT) is characterized by the transition
from hydrated random coils to a regular, non-random struc-
ture that has been described as a b-spiral or a b-sheet. As for
PNIPAAM, this folded and associated state is derived by
dehydration and hydrophobic association of the chains [73].
Strikingly different properties are observed depending on the
length of the repeating pentapeptide and on the nature of
the guest residue Xaa, for example, the presence of a hydro-
phobic amino acid such as tyrosine, shifts the phase transition
to temperatures well below physiological temperature,
whereas the opposite trend is observed in the presence of a
charged amino acid such as glutamic acid [72,74].
The potential of ELPs in cancer therapy has been demon-
strated, particularly in combination with local hyperthermia
of solid tumors. Meyer et al. designed ELPs having an inverse
transition temperature (Tt) of 41 C, by including Val, Gly
and Ala in a 5:3:2 ratio, respectively, at the fourth position
1676 Expert Opin. Drug Deliv. (2013) 10(12)
of the pentapeptide. Heating the tumors above Tt caused
the ELPs to undergo phase transition and aggregation, leading
to approximately twofold increase of the accumulation in the
tumors, as compared to non-heated tumors [75]. Based on the
same principle, a number of authors have thereafter designed
a variety of ELPs as potential drug carriers in anticancer ther-
apy. An ELP-based delivery vehicle containing cell penetrat-
ing peptides derived from the HIV-1 tat protein (Tat) was
conjugated to the cytotoxic DOX. The results showed that
cellular internalization in uterine sarcoma cells was enhanced
by the presence of Tat and by the hyperthermia treatment.
Expressively, the cytotoxicity was enhanced 20-fold following
hyperthermia treatment [76]. Encouraging results were
observed in subsequent studies, where the ELP--DOX conju-
gates were shown to overcome drug resistance in multidrug
resistant human carcinoma cells, particularly after the thermal
treatment [77]. In this study, the authors used a drug-resistant
cell line, characterized by a high expression of the membrane
transporter P-glycoprotein (P-gp), which is a well-known
drug efflux pump for xenobiotics. Immunofluorescence and
flow cytometry experiments confirmed that low intra-cellular
DOX levels were associated with a high P-gp expression when
free DOX was used. In contrast, the ELP--DOX conjugates
accumulated in all cells equally, regardless of the P-gp expres-
sion, suggesting that they were able to overcome cellular efflux
by P-gp. A hyperthermia protocol led to increased toxicity and
apoptosis due to thermally induced aggregation of the
conjugates [77].
Optimization studies have additionally shown that by
employing a thermal cycling protocol -- alternating between
cycles of hyperthermia and normothermia -- the accumulation
of ELP drug carriers in tumors can be significantly
improved [78]. Further, when designing ELP--drug conjugates,
shorter linkers seem preferable in order to achieve faster tran-
sition kinetics and faster drug release following linker cleav-
age [79]. Recently, ELP block copolymers comprising Asp- or
Glu-based blocks were designed for facile conjugation with
the hydrophobic drug geldanamycin with applications in
thermo-targeting chemotherapy. Because of the high hydro-
phobicity of the drug conjugate, it was suggested that the con-
jugate self-assembled into core-shell micelles, where the drug
could be shielded in the core by the hydrated ELP shell.
The transition temperature was found to be dependent on
polymer MW and on the pH of the medium, while drug
release was faster at low pH and high temperature [80].
3. Temperature-responsive hydrogels
In drug delivery, in situ gelling systems are a particularly
appealing concept. By using temperature-responsive poly-
mers, one can for instance produce a low viscosity solution
that can easily be injected but that undergoes fast gelation
in situ as a response to body temperature. Once formed, the
hydrogels can provide the drug with protection from the sur-
rounding medium, and may also be used as drug carriers.
 Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action
Importantly, the properties of ‘smart’ hydrogels can be
employed to control the loading and release of encapsulated
drugs as a response to changes in the surrounding
environment such as temperature [81,82].
3.1 Hydrogels based on PEO/PPO copolymers
Concentrated Pluronic solutions are a well-known example of
temperature-responsive gels [18,60,83]. It has been hypothesized
that at sufficiently high concentrations, the micelles arrange in
a packed, entangled structure (such as cubic crystalline phase),
creating the three dimensional structure that constitutes the
gel [63]. As in the micellization process, this sol-gel transition
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also depends on the hydrophilic--hydrophobic balance and
on polymer concentration. Under specific conditions, the
transition can take place close to physiological temperature,
allowing in situ gelation [18]. At even higher temperatures,
the gel becomes an opaque solution (cloud point), due to
the shrinkage of the PEO corona and higher interactions
with the PPO core [84].
Pluronic gels are characterized by a weak mechanical
strength, short residence time and high permeability, which
limit their applicability in drug delivery [82,85]. Stabilization
can however be promoted by cross-linking the Pluronic gels,
resulting in a controlled drug release [86]. A similar effect can
For personal use only.
also be attained by chemically modifying the copolymer
composition or its structural properties such as the MW.
Xiong et al. grafted two short PCL blocks [87] -- or alternatively
two hydrophobic PLA blocks [88] -- to both ends of an
F87 Pluronic copolymer, and observed that the thermorespon-
sive properties of Pluronic were retained after the modifica-
tion, even though the critical micellization temperature
decreased due to the higher hydrophobicity of the pentablock
copolymer. A low burst release of a model drug was also dem-
onstrated, suggesting an enhanced degree of stabilization of the
hydrogel [87,88]. Another very interesting strategy is based on
the bulk polymerization of Pluronic F127 into high MW
copolymers. In this case, an increase in the degree of polymer-
ization caused a decrease in the gel point and an increase in gel
viscosity, with the drug retention capacity of the gel increasing
up to five times compared to the corresponding native
F127 aqueous solution [89]. High MW multi-block Pluronics
coupled by degradable ester moieties show a decrease in the
gel concentration and an enhanced stability, with gel durations
increasing from 8 h to several weeks, as the MW is increased
from 4000 to 40,000 g mol--1 [90]. Transparent gels exhibiting
rapid thermoresponsive gelation at remarkably low polymer
concentrations (0.5 wt%) can be produced by grafting poly
(acrylic acid) onto the PEO--PPO--PEO backbone [91,92].
In contrast to Pluronics, Poloxamines are still scarcely inves-
tigated as hydrogels and only a small number of reports can be
found in the literature concerning potential applications,
structural versatility and properties, and introduction of chem-
ical modifications on the copolymer. Future experiments will
probably reveal new applications for Poloxamines, due to their
characteristic X-shape and presence of a central ethyleneimine
Expert Opin. Drug Deliv. (2013) 10(12)
group. So far, the interest of these in situ gelling systems has
been demonstrated in tissue engineering applications [67].
3.2 Hydrogels based on PEG/PLGA copolymers
PEG-b--PLGA-b--PEG was designed for an improved durabil-
ity and biocompatibility [84,93,94]. As in the case of Pluronics, a
sol-gel transition can be observed when the temperature of the
solution is increased. The conjecture is that micellar growth
and polymer--polymer interactions account for the thermores-
ponsive gelation of the copolymer [93]. These micelles are
formed by a PLGA (hydrophobic) core, and a PEG (hydro-
philic) corona. As the temperature is increased, the micelles
associate to form the interconnected network that constitutes
the gel. At sufficiently high temperatures, the hydrogen bonds
are broken and the reduced hydration of the polymer leads to
a high degree of aggregation and to macroscopic phase-
separation [95]. Longer PLGA blocks shift the sol-gel transi-
tion to lower polymer concentrations while the gel tempera-
ture region is also widened. The presence of a longer
hydrophobic block has been suggested to form larger micelles,
which tend to pack closely at lower concentrations, increasing
the stability of the network. This effect is even more evident
for PLGA blocks containing a higher amount of D,L-lactide,
which contributes to increase the hydrophobicity of the
copolymer. Conversely, longer PEG blocks can shift the gel
temperature to higher values [93]. These results emphasize
that the composition and MW of the copolymers can have a
pronounced effect on the gel properties, and should therefore
be controlled in order to meet the specific requirements of the
envisioned applications. PEG-PLGA--PEG copolymers were
shown to have a low viscosity at low temperatures and to
undergo rapid viscosification at temperatures > 25 C. When
the solution was injected subcutaneously into rats, the gels
persisted for more than a month, which markedly contrasted
with the low stability of the Pluronic gels, suggesting that dif-
ferent gel degradation mechanisms are involved [96]. More-
over, the gels have shown to be promising drug delivery
systems, with higher drug retention being observed for hydro-
phobic drugs that are partitioned into the PLGA micelle
cores. Longer PLGA blocks contribute to retain higher
amounts of hydrophobic drugs and to slow down their
release. This is further accentuated by using higher polymer
concentrations [97]. Studies on PLGA-graft-PEG [98] and
PEG-graft-PLGA [99] revealed the retention of thermorespon-
sive properties, suggesting that the polymer architecture is not
decisive for the gelation of the copolymer. However, copoly-
mers with a hydrophilic (PEG) backbone have a short-term
durability, being more adequate for short-term drug release,
whereas copolymers with hydrophobic (PLGA) backbones
can last for significantly longer periods of time [98,99].
3.3PNIPAAM-based hydrogels
PNIPAAM hydrogels can be prepared for instance by
chemical cross-linking with a cross-linker agent such as N,
N¢-methylenebis(acrylamide) [100-102]. Below the LCST, the
1677
 M. T. Calejo et al.
hydrophilic amide groups of the polymer form hydrogen
bonds with the surrounding water molecules and the network
swells to create a gel. As the temperature is increased above the
LCST, the hydrogen bonds are disrupted and hydrophobic
interactions between the hydrophobic isopropyl groups cause
aggregation and collapse into a stiff and opaque struc-
ture [100,101]. This phase-transition behavior is reversible, and
decreasing the temperature below the LCST will therefore
result in the hydration and re-swelling of the network [100,101].
The level of cross-linking of PNIPAAM gels can influence
the porosity and release rates of loaded drugs [102]. Further-
more, the properties of the hydrogels can be changed by
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copolymerization of NIPAAM with other monomers.
Copolymerization with a hydrophilic monomer, for instance,
can induce the formation of a macroporous structure, due to
the enhanced swelling and de-swelling properties of the
polymer [103].
Self-gelling systems have been produced by synthesizing
copolymers of PNIPAAM with natural polymers such as hya-
luronic acid [104], alginate [105] and chitosan [106]. Copolymer-
ization can be used to enhance the mechanical strength of the
hydrogel and to prevent syneresis (expulsion of water from the
gel), as shown for PNIPAAM-g-methylcellulose [107], or to
increase the biodegradability of the copolymer, as presented
For personal use only.
for gelatin-g-PNIPAAM [11]. Comb-type structures also con-
tribute to induce a fast transition due to the high mobility
of the temperature-responsive PNIPAAM grafted to the chain
end [104].
An instructive study concerning the importance of polymer
architecture on hydrogel properties was carried out by Lin &
Cheng, using block or star copolymers with a hydrophilic
PEG segment and PNIPAAM terminal segments [58]. The
results from this work revealed that the sol-gel transition in
AB block copolymers is probably due to close micelle packing,
whereas the presence of multiple arms in star copolymers can
enable gel formation via a physical cross-linking mechanism.
The rheological properties of the gels were strongly dependent
on the copolymer architecture. Gels formed by the 4-arm
copolymer were stronger and more deformable than those
produced by the 2- and 8-arm counterparts, indicating the
importance of the number of arms in network formation.
The soft gels of the 1 arm copolymer are consistent with the
higher mobility and deformability of the PEG blocks com-
pared to multi-arm copolymers. Increasing the number of
arms beyond a certain limit was suggested to hinder the
inter-molecular association between the PNIPAAM blocks,
again resulting in weaker gels [58].
4.Temperature-responsive polymers as gene
delivery vectors
In gene therapy, a vector containing nucleic acids is intro-
duced into target cells so as to modify gene expression in a
way that prevents, stops or reverses a pathology [108].
1678 Expert Opin. Drug Deliv. (2013) 10(12)
Viral vectors have shown to be the most efficient carriers
for nucleic acids, particularly DNA [109,110], but a number of
serious side effects have limited their clinical use. An excessive
immune response is still nowadays one of the major hurdles of
gene therapy. The presence of viral capsid proteins can trigger
the activation of the immunological defense systems, particu-
larly in cases where the patient has been sensitized by wild-
type infections [111]. The occurrence of acute inflammatory
response and delayed humoral or cellular immune responses
also limit the possibility of repeated administrations. Leukae-
mia cases have been reported, due to random integration of
the genes into the host’s chromosome [112]. Furthermore, the
production procedure is quite complex, and the viruses are
difficult to produce on a large-scale [109].
As a general rule, water-soluble polymers used in drug
delivery have minimal immunogenicity and antigenicity,
with the specific immune response depending on the physico-
chemical characteristics of the polymer, for example, compo-
sition, size, shape and electric charge [113,114]. Their generally
low immunogenicity and toxicity make them interesting alter-
natives to the viral carriers [109,112]. In addition, the large scale
production is considered relatively easy, and the non-viral vec-
tors can be especially designed to target specific cells or tissues.
The major limitation of the non-viral vectors is that they are
usually less efficient than viruses. In spite of this, the carriers
can be administered repeatedly, if necessary, and the methods
can be changed to improve the gene expression [112].
Two of the most important requirements of a gene carrier
are that it forms tight complexes with the DNA (protecting
it from the degradation by nucleases, condensing it to an
appropriate size and endowing it with a positive charge suit-
able for internalization) and that, once in the nucleus, it disso-
ciates from the payload so that gene expression may take place
(Figure 3). The use of stimuli-responsive polymers is consid-
ered particularly advantageous since by applying a physical
stimulus, such as light and temperature, complex association
or dissociation can be controlled. In addition, one can control
the site, timing and duration of the gene expression [115].
Thermoresponsive polymers are unusually versatile vectors
in the sense that the association and dissociation of the poly-
mer--polynucleotide complexes can be promoted by simply
changing the temperature. Copolymers with a LCST below
body temperature can condense the DNA to an appropriate
size for cell internalization at higher temperatures, while dis-
sociation of the complexes in the target cell can be promoted
by decreasing the temperature (Figure 4). An enhanced gene
expression may thus be observed, as demonstrated by studies
on PNIPAAM copolymers [116-118].
In a gene delivery study, a block copolymer containing
PNIPAAM and the positively charged PDMAEMA was
used [119]. The presence of positively charged units facilitates
the electrostatic interactions with the negatively charged phos-
phate groups of DNA, thereby leading to the formation of
polymer--DNA complexes (polyplexes). The copolymer was
able to condense the DNA into small particles, while the
 Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action

Microtubu
le

DNA DNA
release 1 release 2 DNA
release 3
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DNA binding and Penetration Cellular Intracellular Endosomal Nuclear Transcriptional

colloidal stability of ECM uptake trafficking escape translocation processing

Figure 3. After shuttling a DNA payload into the cell, a carrier must then release it. This happens either i) in the endocytic
vesicle, ii) in the cytoplasm or iii) in the nucleus. Ideally, the carrier would switch from a protective element to one favouring
release at one of these locations.
Taken from [16] with permission.
For personal use only.

+
+
+ + + +
+ +
+ +
+ + +
+ + +
+ T > LCST T < LCST
+

Figure 4. Use of cationic thermoresponsive copolymers in gene delivery. At low temperatures, the copolymer is found in an
extended, hydrated conformation, facilitating the establishment of electrostatic interactions with the negatively charged
DNA (forming the polyplexes). As the temperature is increased to values higher than the LCST, a coil-to-globule transition
takes place, encapsulating DNA and protecting it from the external environment. The positively charged nanoparticles can
interact with the negatively charged cell surface, facilitating endocytosis. Once the polyplexes are found in the intra-cellular
compartment, an externally applied cooling procedure contributes to dissociate the polyplexes; thereby releasing the DNA.

overall presence of positive charges on the complexes was
believed to facilitate cell uptake, leading to higher transfection
efficiencies. Interestingly, an increase in NIPAAM content led
to a decrease in the charge density, indicating that the ther-
moresponsive block somehow was able to cover the positively
charged groups of the copolymer. This resulted in a decreased
cytotoxicity, but also in a lower transfection efficiency due to
a lower ability of the complexes to interact with the oppositely
charged cell membranes [119].
Polymers that respond to several stimuli may turn up to be
particularly promising gene carriers, due to their enhanced
capacity to overcome physiological barriers. Copolymeriza-
tion of PNIPAAM with PEI, for instance, provides the gene
carrier with both temperature- and pH-sensitivity [120]. PEI
is a well-known polycation, recognized for its ability to escape
the endosomes due to its high protonation capacity, creating
the so-called ‘proton sponge’ effect. PNIPAAM-b-PEI
copolymers have shown a high DNA condensation capacity
at body temperature, leading to successful gene expression.
Properties of the complexes (size and charge density), DNA
uptake and gene expression are however strongly dependent
on the MW of the copolymer and on the architecture of
PEI [120].
Zintchenko et al. [121] prepared PNIPAAM-b-PEI copoly-
mers containing hydrophilic monomers, namely acrylamide
(AAm) or vinylpyrrolidinone (VP) that allowed the

Expert Opin. Drug Deliv. (2013) 10(12) 1679

 M. T. Calejo et al.
adjustment of the LCST to temperatures higher than 32 C.
By changing the copolymer composition, a range of copoly-
mers was prepared, with transition temperatures between
37 and 42 C. At body temperature, these copolymers formed
complexes with DNA, shielding it from unspecific interac-
tions with the surrounding media. The complexes were small
but aggregated significantly when incubated at 42 C because
of the thermoresponsive behavior of PNIPAAM, which led
to an increase in gene expression. It was suggested that the for-
mation of large aggregates facilitated the endosomal
release [121]. Studies on cancer therapy applications were sub-
sequently performed, using a thermoresponsive copolymer
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based on VP and NIPAAM grafted on PEI (LCST 42 C) as
a DNA carrier [122]. By employing local hyperthermia to
tumor tissues, the accumulation and aggregation of the com-
plexes in the tumor was remarkably increased and an
improved level of transgene expression was observed, confirm-
ing tissue selectivity [122].
The importance of molecular architecture in binding and
release of DNA was demonstrated using branched PEI with
grafted PNIPAAM chains [123]. Copolymers prepared with
low MW PNIPAAM grafts (LCST 40 -- 44 C) were more
efficient gene carriers than copolymers with high MW grafts
(LCST to 34 -- 37 C), owing to alterations in structure and
For personal use only.
architecture of the complexes. The lower transition tempera-
ture of the copolymer containing the high MW PNIPAAM
made it particularly sensitive to temperature alterations.
Gene expression could be considerably incremented when
the complexes were incubated above the LCST, leading to
DNA compaction, and when a cooling step was subsequently
employed, prompting the dissociation of the complexes and
enabling DNA release. The relaxation of the polyplex archi-
tecture by the temperature decrease was further suggested to
facilitate the endosomal escape by the ‘proton sponge’
effect [123]. Studies on other PNIPAAM copolymers, namely
chitosan-NIPAAM/vinyl laurate [124] and N,N,N-trimethyl
chitosan chloride-g-PNIPAAM [125] similarly confirmed the
potential of using temperature protocols to trigger the associa-
tion--dissociation of the complex. In the latter, trimethyl chi-
tosan was used instead of the native collagen, attributable to
its higher solubility and superior capacity to open tight junc-
tions of cells (increased paracellular permeability), whereas the
presence of PNIPAAM improved the biocompatibility of the
vector [125]. In this context, it is clear that multiple strategies
can be simultaneously employed to improve the gene delivery
efficiency with minimal toxicological implications, and that
this can be achieved by modifying polymer architecture
and composition.
One of the most critical properties of a thermoresponsive
polymer is its hydrophilic-hydrophobic balance, since this
can have a dramatic effect on the structures of the polyplexes
and on the gene delivery efficiency of the carrier. This has
been shown in a recent report, where cationic block copoly-
mers based on PNIPAAM-b-poly((3-acrylamidopropyl)tri-
methylammonium chloride) (PNIPAAM-b-PAMPTMA(+))
1680 Expert Opin. Drug Deliv. (2013) 10(12)
were studied [126]. Copolymers with short charged blocks
were highly associative at 37 C, forming compact core-shell
structures that were able to protect the DNA from the exter-
nal environment and led to expressive transfection efficiencies.
However, for a constant length of PNIPAAM, increasing the
length of the charged PAMPTMA led to an increased degree
of charge repulsion between the polymer molecules, obstruct-
ing to some extent the thermoresponsive association of PNI-
PAAM with the resulting formation of polyplexes with an
‘open’ structure. The low compactness of these polyplexes
was ascertained as the key factor for the lower transfection
efficiency, supposedly due to a lower capacity of the carrier
to destabilize the lipid bilayers. For long charged blocks, a
very long PNIPAAM block was therefore found essential in
order to ensure the formation of compact nanoparticles that
lead to a successful gene delivery [126].
In an innovative concept, Choi et al. [127]. developed cross-
linked Pluronic/PEI nanocapsules, alternating between a
collapsed and shrunken state at higher temperatures and a
swollen state at lower temperatures [127]. In a subsequent
study, the nanocapsules were evaluated as carriers for small
interfering RNA (siRNA), with enhanced delivery to the cyto-
sol. Shrunken siRNA-nanocapsules were taken up by endocy-
tosis and captured by the endosomes. A cold shock treatment
applied to cells caused the nanocapsules to dramatically
increase their size (swollen state), causing the physical disrup-
tion of the endosomal membrane. As a consequence, the com-
plexes were released from the endosomes, thereby avoiding
the digestion of the siRNA, and leading to the envisioned
gene silencing effect [128].
5. Conclusion
The application of responsive ‘smart’ polymers in drug deliv-
ery and gene transfer has grown tremendously in recent years,
and this has attracted scientists over a broad interdisciplinary
research field. The use of responsive polymers as nano-
particles and hydrogel drug delivery systems constitutes an
interesting approach to improve the efficiency of the delivery
of drugs and transfer of genes.
In this review, the importance of the interplay between the
hydrophobic and hydrophilic interactions, as well as the
power of Coulomb forces, on the structure of temperature-
responsive copolymers in aqueous solution was demonstrated
by using several examples from the literature. A number of
studies have shown that the thermoresponsive properties of
the drug delivery system -- significantly affected by polymer
structure and composition -- can be used not only to facilitate
drug loading, but also to target specific cell types and com-
partments. In vivo studies are still scarce, but drug targeting
to internal organs may be achieved by local hypo/hyperther-
mia, for example, using a catheter, prompting the destabiliza-
tion of the carrier and releasing the drug at the intended site.
As drug delivery vehicles, thermoresponsive hydrogels are
 Thermoresponsive polymers as gene and drug delivery vectors: architecture and mechanism of action
particularly interesting systems that can form depot formula-
tions in situ after local injection, as a response to body
temperature.
In this overview it was demonstrated that polymer architec-
ture, composition of hydrophilic and hydrophobic sequences,
and MW are key elements for the mechanisms of action of
systems in drug and gene delivery applications. The stealth
effect of nano-carriers covered with hydrophilic shells prevent-
ing uptake by macrophages is examined, and it is discussed
how the low MW of the polymer may allow easy elimination
by glomerular filtration, thereby avoiding accumulation in the
organism. The power of polymer composition on vital issues
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Oslo universitetssykehus Aker on 12/01/13
such as biocompatibility, biodegradability and toxicity is
also evident. The studies presented here have demonstrated
that there are still many challenges in this field to develop
more efficient polymer-based carriers for gene and drug
delivery applications.
6. Expert opinion
Conventional drug therapy for most diseases of humans
involves flooding the body with drugs, with many undesirable
side effects. An attractive concept has emerged over the past
few years of enclosing drugs within biocompatible and
For personal use only.
biodegradable responsive polymers or liposomes in the form
of a nanoparticle, the physical chemical properties of which
can be tailor-made to enclose drugs of diverse nature.
A broad range of different materials are being tested towards
this goal, worldwide. Both self-assembled and chemically
cross-linked particles can be made that encapsulate water-
soluble or poorly water-soluble drugs and release them in a
sustained manner in cells of interest. Such particles prevent
the drug from interacting prematurely within the body upon
administration. In addition, specific molecules on the particle
surface can be used to target nano-structures to cells of inter-
est, such as cancer cells or infected cells, enriched in receptors
that recognize the targeting molecules. These cells can then
take up the particles by endocytic mechanisms, which deliver
the particles to the lysosomes where the particles are gradually
degraded, resulting in a slow, steady release of the drug in the
cells. Polymers and especially responsive polymer carriers con-
stitute an important platform for drug delivery and gene
transfer applications. A vast arsenal of sophisticated polymeric
nano-carriers has been developed in recent years with great
Expert Opin. Drug Deliv. (2013) 10(12)
potential for site-specific drug delivery applications. In spite
of the advanced responsive self-assembled structures, there is
still a severe lack of understanding of how factors such as poly-
mer architecture, MW, composition of copolymers, hydro-
philic--hydrophobic balance, size and size distribution of the
carriers will affect the loading efficiency and drug release.
A thorough knowledge of these factors is necessary to be
able to develop optimal nano-carriers for the future. Another
issue that is not fully understood is how small the carriers
need to be to easily be eliminated by glomerular filtration
and thereby avoid accumulation of polymer in the organism.
Further, it is known that the circulation time in the body for
polymer carries that are decorated with a layer of PEG is pro-
longed, but it has not been established how thick the PEG
layer needs to be and we do not know if the stealth effect
also works with other hydrophilic polymers. The conventional
procedures that are employed today to prepare nano-carriers
are nano-precipitation and the double emulsion method,
but they are not optimal for the manufacturing of particles
with controllable sizes and narrow size distribution. The use
of micro-fluidic and stopped-flow devices in the preparation
of nano-carriers is more promising when it comes to the con-
trol of size, size distribution, and to achieve a high loading
capacity of the drug. A great challenge in the future is to
design well-regulated responsive carrier structures that can
encapsulate drugs of different hydrophobicity. It is also a vital
task in the years to come to develop efficient receptors
attached on the targeting molecules so that they are specifi-
cally designed to attack cancer cells and not healthy cells.
When it comes to gene delivery, some auspicious responsive
carrier structures have already been designed from copoly-
mers, but more sophisticated and tailor-made carriers are yet
to come.
It seems that in the future, specially designed functional
polymers with specific features will play a crucial role in the
development of carriers for drug and gene, and more sophis-
ticated ‘smart’ polymers of various structures that respond to
different stimuli will appear for clinical tests.
Declaration of interest
The authors declare no conflict of interest. The authors are
grateful for financial support from the Norwegian Research
Council (Project Number 190403).
1681
 M. T. Calejo et al.
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Affiliation
Maria Teresa Calejo1,2, Sverre Arne Sande1 &
Bo Nystr€om†3
For personal use only.

†
Author for correspondence
1
University of Oslo, School of Pharmacy,
Department of Pharmaceutics,
P.O. Box 1068, Blindern, N-0316 Oslo, Norway
2
Tampere University of Technology,
Department of Electronics and
Communications Engineering, Finland
3
University of Oslo, Department of Chemistry,
P.O. Box 1033 Blindern N-0315 Oslo, Norway
E-mail: bo.nystrom@kjemi.uio.no

1686 Expert Opin. Drug Deliv. (2013) 10(12)