Future

Review

Medicinal
Chemistry

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Molecularly imprinted polymeric microand nano-particles for the targeted

delivery of active molecules

Molecular imprinting (MI) represents a strategy to introduce a molecular memory

in a polymeric system obtaining materials with specific recognition properties. MI
particles can be used as drug delivery systems providing a targeted release and thus
reducing the side effects. The introduction of molecular recognition properties on a
polymeric drug carrier represents a challenge in the development of targeted delivery
systems to increase their efficiency. This review will summarize the limited number
of drug delivery MI particles described in the literature along with an overview of
potential solutions for a larger exploitation of MI particles as targeted drug delivery
carriers. Molecularly imprinted drug carriers can be considered interesting candidates
to significantly improve the efficiency of a controlled drug treatment.

Molecular imprinting technology:

what can this technique do?
The story of molecular imprinting (MI) applied
to organic polymers has been started when, for
the first time, Wulff and Sarhan[1] described a
polymeric system with an enzyme-like behavior able to separate racemic mixtures of components. In this communication, they referred
to porous and swellable polymeric matrices
containing functional sites able to retain different enantiomers but the concept of the
molecular imprinting was not yet completely
introduced. After ten years, Arshady and Mosbach published a communication [2] describing
a substrate-specific polymer obtained through
a hostguest polymerization that was introduced as an alternative of the conventional
template polymerization. This novel
procedure was described as simply a mixing
procedure, and no chemical attachment to the
monomeric units is required. The first paper
was the starting point for the covalent MI
approach applied to polymers while the second
for the noncovalent one. Nowadays, Scopus
reports about 8000 papers related to the MI
technology on organic polymers and about
3000 in the last 5 years.
The molecular imprinting technology
is a synthetic way to obtain cross-linked

10.4155/FMC.14.140 2015 Future Science Ltd

Mariacristina Gagliardi*,1
& Barbara Mazzolai1
Center for Micro Bio-Robotics @ SSSA,
Istituto Italiano di Tecnologia, viale
Rinaldo Piaggio 34, 56025, Pontedera,
Pisa, Italy
*Author for correspondence:
mariacristina.gagliardi@iit.it

polymers with molecular recognition capabilities toward a template molecule introduced in the reaction during the synthesis. A
conventional method to obtain a MI polymer
is in liquid phase and can be schematized in
four steps[3] (Figure 1) : Figure 1A shows in
solution the template molecule and the
functional monomers are well dissolved
in the reaction medium; Figure 1B shows
complexation (or self-assembly) monomer
functionalities interact with the template
molecule forming a stable chemical complex, characterized by covalent or noncovalent interactions, after a self-assembling
procedure; Figure 1C shows locking functional monomers are cross-linked around the
template molecule and frozen in the position assumed during the complexation step;
Figure 1D shows extraction the template
molecule is extracted from the cross-linked
matrix leaving functional cavities into the
formed polymer; functionalized sites are
complementary to the template in terms
of shape and position of functional groups
involved in the complexation process.
As previously mentioned, there are two
main approaches to obtain complexes
between template and functional monomers: covalent and noncovalent. These two

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Figure 1. Steps of a conventional noncovalent

molecular imprinting synthesis. (A) In solution,
(B) complexation, (C) locking and (D) extraction.

approaches differ in the type of chemical bonds arising during the formation of the complexes. In the case
of the covalent approach, chemical bonds result which
are difficult to cleave during the extraction phase; in
the noncovalent approach, bonds are electrostatic,
hydrogen-bonds or ionic and the template can be easily
extracted through a simple washing procedure.
Considering that more stable complexes give higher
molecular recognition, the covalent approach seems to
be the better way to increase the performance of a MI
system. Covalent bonds between functional monomers
and template may be reversible or cleavable through
a simple chemical procedure. Obtained molecularly
imprinted polymers can recognize the template molecule used for the preparation after the extraction in
two different ways: formation of covalent bonds after a
chemical reaction; or formation of noncovalent bonds
(semicovalent approach). The recognition based on
the formation of covalent bonds between template and
MI polymer in rebinding tests was the first case investigated in the literature [1,4] . The main advantage of
this approach is represented by the stoichiometric ratio
between monomer and molecule to be recognized and
the homogeneous distribution of the recognition sites
in the polymeric structure. Even if recognition sites
obtained via covalent imprinting are more efficient than
those obtained through the noncovalent procedure, the
binding kinetics may be slow, therefore making this
route impractical for several applications. In late years,
in order to speed up the binding kinetics without the
loss of control of the topochemistry in the smart
cavities, the concept of the covalent MI was refined and

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a novel approach, based on the use of partially cleavable

templates, was developed. In this case, part of the template molecule is removed leaving functional groups in
the cavities. In the work of Whitcombe et al. [5] this
approach allowed the preparation of polymeric receptors for cholesterol. As demonstrated, the template molecule (4-vinylphenyl carbonate ester) was covalently
bound to the polymer during the MI polymer formation and then removed by hydrolysis, producing CO2.
After the hydrolytic cleavage of the template, phenolic
residues remained on the polymer matrix constituting
sites for the smart interactions with the molecule of
interest (cholesterol). The literature also reports mixed
noncovalent/semicovalent approaches for the preparation of polymers with molecular recognition toward
vanillin [6] and other substrates [7] .
The covalent approach for the synthesis of MI polymers shows some additional limitations. This procedure
can lead to the formation of too stable complexes with
the impossibility to extract the template molecule from
the matrix. In addition, the number of useful monomers containing polymerizable functionalities or that
can be derivatized is limited. Furthermore the kind of
interactions that can be obtained is restricted: chemical
reactions may occur only in the presence of the molecule being bonded and without any catalyst or initiator.
It means that there are few reactions that are thermodynamically possible in this condition and the kinetics
may be too slow for it to be useful for any process.
The presence of strong interactions between template and monomers in the noncovalent approach are
the premise to obtain a well-working MI polymer. Thus
the self-assembly is an important factor that can predict
the performance of the final system in terms of recognition properties [810] . Computational simulations of
interactions between template molecule and functional
monomers represent an important step in the optimization of the final product [11] . The quantification of these
interactions and a preview of the final topochemistry of
the MI cavities are the major results of a computational
study then a prescreening to evaluate the best monomers can be done via this study. The problem can be
approached with the force-field method [12] , molecular dynamics [13,14] or more elaborate theories such
as the Mulliken charge method [1518] , the molecular
orbital theories (e.g., the HartreeFock[1923], Moller
Plesset [19,2425]) and the electronic band structure
theories (e.g., the density functional theory [2629]).
The previous discussion is a general overview of the
MI technology and it is valid for monoliths, gels and particles. Recognition performances can be significantly
increased by varying the geometry and the shape of the
system. In the case of particles, this particular structure has a maximized surface-to-volume ratio and it is

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Molecularly imprinted polymeric micro- & nano-particles

an important characteristic in terms of exposed surface

of the cavities that result in greater accessibility of the
template. This aspect is fundamental for the binding
capacity and kinetics.
In a traditional scheme, MI particles (MIPs) are prepared in the form of monolithic nanogel that is ground
after the synthesis to obtain the particles. However,
this procedure leads to the formation of irregularly
shaped particles and the efficiency of the recognition properties may be affected by the nonuniform
dimensions. For this reason, more efficient synthetic
techniques were employed with better results. Two
common synthetic routes were applied in the preparation of MI particles with controlled size and shape:
suspension polymerization [3033] and precipitation
polymerization [3436] . Even if these strategies are not
straightforward as the synthesis of a monolith, there
are several benefits. Yeetal. [30] have demonstrated the
effect of the suspension polymerization on the particle
morphology compared with the conventional synthesis. Size and shape of obtained microparticles were controlled and recognition properties resulted better when
compared with the analogous nanoparticles obtained
by the monolith ground. This polymerization scheme
allowed obtaining pH-sensitive MI microspheres
capable of selectively binding the template on the basis
of ionic interactions that were weakened during the
polymerization in the presence of water in the reaction medium, as demonstrated by Lai et al. [31] . It was
reported that the suspension polymerization increased
not only the yield of reaction and the uniformity of
microparticles dimensions but also the sorbent capacity of the polymer [32] . Recent advances [33] have demonstrated that MI microspheres can be controlled in
size and shape by varying the stirring rate, the reaction
medium composition and the dispersant.
Good results were also obtained by applying the
precipitation polymerization scheme. Results of a precipitation MI polymerization with the conventional
bulk synthesis were compared [35] , showing that the
specific surface area of micropore within microparticles obtained by precipitation was significantly higher
than that obtained by bulk polymerization resulting
in an increase of recognition properties. This reaction
scheme allows tuning the size of obtained MI particles
by varying the size of the cross-linker molecule [36] .
The recent literature also reports the polymerization
of MI polymers through reversible addition-fragmentation chain transfer (RAFT) [3744] and atom-transfer radical-polymerization (ATRP) [4548] . Attempts
to strictly control shape and dimension of produced
particles were made not only by varying the solvent
and the monomer concentration but also by adding
auxiliary templates [49] .

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Other chemical aspects

A large number of conventional applications of the MI

technology are devoted to the preparation of polymers
selective towards small molecules, polymerizing functional monomers able to form hydrogen bonds with
the template in a nonpolar solvent. The use of small
target molecules containing one heteroatom (e.g.,
nitrogen, oxygen, phosphorous or sulfur) is frequent
because this kind of template exerts few polar interactions localized on the heteroatom that becomes the
more relevant site during the formation of the complex.
This is the more common reaction scheme because
small template molecules can interact with the monomers only through one kind of interactions. This condition has a favorable energy in nonpolar solvents, and
same interactions are not favored between monomers
and solvent. Monomers generally used, in particular
for the noncovalent approach, are polar and small molecules, such as acrylic[16] and methacrylic acid [50,51] .
These two monomers are frequently used in MI preparation because they are able to work as both hydrogen
donors and acceptors. Also nitrogen-containing monomers, with amine or amide functional groups, result
particularly useful for MI synthesis as biomimetic
systems. The biomimicry is related to the fact that in
living beings a large number of recognition processes
involve peptides or proteins containing nitrogen-based
moieties.
Functional monomers can produce binding sites
capable of one or more interaction sites while, polymers, on the other hand, can have several sites of
interactions. MI polymers can also be obtained starting from functionalized prepolymers. In this case, a
preformed polymer containing post-functionalizable
(cross-linkable) groups is prepared and then, after the
interaction with the template molecule, crosslinked.
In the work of Matsui et al. [52] a poly(methacrylic
Key terms
Template polymerization: Particular type of
polymerization reaction supported on a preformed polymer
able to influence the chain growth and to confer to the
growing macromolecules some specific physicochemical
properties.
Topochemistry: Study of the chemistry related to
particular reactions involving at least one reactant in solid
form, occurring only at specific sites of the solid; reactions
can be reversible or not and may cause large modifications
in the structure of the solid reactant.
Biomimetic system: In general, the term biomimicry is
referred to the aptitude of a synthetic system to imitate the
behavior of a natural system; in the case of MI polymers,
the biomimicry is narrowed to the physicochemical
interactions that are established between the smart cavities
of the MI polymer and the molecule used as template.

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acid)-based MI matrix with recognition toward
()-cinchonidine was prepared in a hydrophilic
alcohol-based medium. The cited work reported the
effectiveness of the MI system to rebind the template
on the basis of effective MI interactions instead of
polar interactions, that are unfavorable in hydrophilic
environments.
In addition to monomers and the template molecule, the effect of the solvent used for the reaction
is fundamental. The solvent is generally indicated as
porogen because it acts as a factor increasing the porosity of the matrix. Generally, a larger porosity degree
leads to more reachable recognition sites and thus to
better performances. The effect of the solvent was analyzed in literature and it was demonstrated that aprotic
solvents (e.g., toluene, chloroform, dimethyl sulfoxyde
and dimethylformamide) give better results in terms
of specific surface, and thus of rebinding capacity,
than protic ones (e.g., methanol) when polar monomers (e.g., methacrylic acid) are used [53,54] . The effects
of the solvent polarity on porosity were also reported
in other studies [5560] . Use of the Pickering radical
polymerization scheme to increase the MI polymer
porosity has also been reported [6167] . Also polymers
can be used as porogens [37,38] .
Applications of molecularly imprinted
polymers in pharmaceutical & biomedical
fields
Synthetic immunoassays: improved stability
with respect to commercial systems

The capability of MI polymers to selectively recognize

specific molecules allows obtaining high sensitivity
assays for several kinds of analytes. Immunoassays are
commonly used for the detection of a great number of
molecules thanks to their high sensitivity but, because
of their biological nature, they can be variable on the
batch, showing a limited stability and expensive costs.
Synthetic substitutes of the biological assays can overcome these limits and some efforts were made to obtain
them [68] . One of the more common tests used in laboratories that can be emulated with MI polymers is the
ELISA for the quantification of molecules like epinephrine [69] , estrone [70] , olaquindox [71] , atrazine[72] ,
2,4-dichlorophenoxyacetic acid [73] . Nanoparticles
imprinted against the molecule to be quantified are
generally used to coat microplate wells. ELISA assays
based on MI particles have been significantly improved
in the recent years and, as reported in the work of
Chianella etal. [74] , the significantly low detection
limit (2.5pM), the high sensitivity (three orders of
magnitude better than the antibody-based ELISA) and
stability at room temperature for a long time made the
assay particularly interesting to replace the biological

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immunoassays. Attempts to combine two functionalities of MI particles, recognition and signaling, were
reported in the work of Shutov et al. [75] where the
preparation of MI composite magnetic core/shell
nanoparticles (Fe3O4/MI polymer) were developed to
obtain a biomimetic assay.
Sensors & biosensors: label-free detection

The ability of MI polymers to selectively recognize specific molecules has been exploited to obtain label-free
molecular sensors. Three different noteworthy applications reported in the literature are: luminescence,
thermometric and electrochemical sensors.
Luminescence MI sensors are MI polymers containing fluorescent moieties which respond to the binding
events with significant fluorescence changes (Figure 2) .
Lieberzeit et al. [76] have reported a polyurethanebased MI system double-imprinted against polycyclic
aromatic hydrocarbons, obtaining a sensor with very
sensitive detection limits (e.g., 30ng/l for pyrene),
underlining also the beneficial effect of the multiple
imprinting. In the work of Nguyen and Ansell [77]
two fluorescent monomers, 6-styrylcoumarin-4-carboxylic acid and 6-vinylcoumarin-4-carboxylic acid,
were synthesized and used to obtain MI polymers
against (-)-ephedrine as template; polymers exhibited a quenching of fluorescence after recognition of
the template and a selectivity for the template over its
enantiomer (+)-ephedrine and other analogues for low
concentrations of ephedrine.
The thermometric detection through MI polymers
is grounded on the quantification of the heat of binding developed during a recognition process. When
the recognition sites bind the complementary molecule a thermal event occurs thus interactions can be
followed without any additional labelling. The first
application in thermometric detection of MI polymers
was proposed by Lettau and co-workers [78] . They
reported the preparation and the characterization of
an MI thermistor device with a combined activity,
molecular recognition and catalysis, obtained through
a covalent approach. The same principle was applied
in the work of Rajkumar et al. [79] to develop an MI
thermistor toward fructosyl valine and in the work of
Athikomrattanakul et al. [80] for the sensing of nitrofurantoin through noncovalent imprinting. Thermosensing by MI polymers is a synthetic evolution of
the common enzymatic thermistor: enzymatic reactions are commonly followed by calorimetric studies
and it consolidates the biomimicry properties of MI
polymers.
The detection limit of MI polymers can be further
improved by combining with electrochemical methods. The incorporation of metals [81] , grafene [82] or

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Membranes: smart separation of single

molecules & tissue engineering

Membrane separation is a well consolidated process based on the passive transport properties of the
membranes that are dependent on the porosity of the
membrane. Generally, membranes used for the separation are able to retain classes of compound basing
on the molecular dimension. Thus, common commercial membranes generally do not allow the separation
of one specific molecule. The preparation of molecule
specific membranes is a challenging issue that can be
tackled by using MI technology. Thus selective MI
membranes can find interesting bioapplications in
extra corporeal purification systems for the recognition
of solutes in the bloodstream. The literature reports
the development of MI membranes able to recognize
uric acid [93] , low density lipoproteins and cholesterol[94,95] as proof of concept of the final application.
Cell-imprinted membranes were also reported for the
retention of bacteria [96,97] .
In the field of tissue engineering, the use of smart
scaffolds able to guide cell proliferation and differentiation is currently an open challenge. The main goal
of tissue engineering is to reproduce the topography of
the physiological extracellular matrix at the nanoscale,
providing to cells an optimal structure for their
arrangement. The literature reports the use of microor nanopatterned scaffolds capable to align growing
cells helping the wound healing, obtained in the form
of membranes [98,99] or fibers [100] by nanoimprinting
soft-lithography. As a further improvement of this
approach, MI scaffolds can be studied to increase the
biomimicry of the device. A functionalized nanoenvironment for cell differentiation in an MI membrane
was also developed by imprinting biocompatible polymers (polydimethylsiloxane) against mature and dedifferentiated chondrocytes [101] , obtaining a biomimicking substrate for inducing stem cell differentiation.
The literature refers to a 2D scaffold for cell differentiation but the technique may be also extended to 3D
structures.

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Addition of the
molecule to be
retained

Dispersion of
fluorescent MIPs

Partially quenched
MIPs after rebinding

Fluorescence

carbon nanotubes [83,84] into MI polymers is a fast

and low-cost method to improve the performances of
the final system in terms of sensitivity. A composite
conductive MI system can be prepared by: depositing
the polymer on the electrode; electropolymerization
of monomers; or loading the conductive material into
the preformed MI system. MI functionalization can
be obtained before or during the preparation of the
composite system. Examples of electrochemical sensors
for drugs [81,83,8588] , glucose [89] , parabens [90] , neurotransmitters [82,91] , alkaloids [83] , pesticides [92] are
reported in literature following this approach.

Review

Decreasing in
concentration of
retained molecule

Wavelength (nm)
Figure 2. Molecular imprinting particles for labelfree detection. Luminescence molecular imprinting
sensors show changes in fluorescence after binding
events; the figure schematizes one possible case in
which fluorescent molecular imprinting particles are
quenched after the rebinding.
MIP: Molecular imprinting particle.

Drug delivery systems

The exploitation of the MI technology in the preparation of drug delivery systems has been introduced
only in recent years, because of the difficulty in optimizing the material preparation for this application.
Synthesis of polymers for drug delivery systems may
be clean, controlled, biocompatible and with high
yield and to combine these aspects with the MI technology is not a simple task. MI polymers in the form
of gels, scaffolds and particles were formulated for
this aim. In the review of Alvarez-Lorenzo and Concheiro [102] the importance of molecularly imprinted
materials as excipients for drug delivery systems was
highlighted, indicating the three main strategies that
these systems may fulfil: rate-programmed, activation-modulated or feedback-regulated drug delivery
(Figure 3) . Rate-programmed drug delivery systems
are a class of device able to control the drug diffusion outward from the system with a specific kinetic
profile; these systems are able to furnish high delivery rates or prolonged treatments depending on the
delivered drug and the therapeutic treatment. Activation-modulated drug delivery systems are devices in
which the delivery starts after a physical, chemical or
biochemical stimulus, such as the binding of a specific molecule or a chemical variation of the environment. The feedback-regulated drug delivery systems
are triggered by a specific molecule (a chemical or
biochemical compound) having a blood or plasma
concentration dependent on the drug itself.

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Imprinted

Released drug fraction

Released drug fraction

Not imprinted
Time

Time

Released drug fraction

Time
Figure 3. Molecular imprinting particles for controlled drug delivery. (A) Rate-programmed drug delivery: thanks
to the recognition properties of molecular imprinting particles, the release of the active molecule is retarded
and thus a more prolonged drug release than conventional particles is obtained; (B) activation-modulated drug
delivery: the release of the active principle starts when molecular imprinting particles bind a specific molecule;
(C)feedback-regulated drug delivery: the release of the drug is governed by the presence/concentration of a
specific molecule regulated by the pharmacological activity of the released drug.

The use of polymers with smart recognition properties are also interesting in that the activities of the
drugs are significantly influenced by molecular recognition phenomena. The biological environment is
generally characterized by a high number of binding
events with large association constants (in the order
of magnitude of 103 1012 l/mol [103]). Optimized MI
systems can reach similar values and then can be
considered interesting candidates for such applications.
The major interest in the use of MI polymers as
drug delivery systems is related to their capability to
attenuate the delivery rate thanks to the high affinity toward the template molecule. Allender et al. [104]
reported a transdermal delivery system composed of
poly(methacrylic acid) cross-linked with EGDMA and
able to release with a controlled kinetics the loaded
drug (propanolol). Obtained systems were capable to
furnish prolonged delivery profiles, in particular in the
case of the system with the lower drug loading. Similar
results were reported in the work of Cai et al. [105] for
the release of tetracycline. In this paper the MI system
(poly(methacrylic acid) cross-linked with EGDMA)

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was capable to release kinetics twice delay with respect

to the non-MIP reference system in the first part of
the release; however, the amount of drug released from
MIP was higher than the amount released from the reference system, thanks to the higher affinity of the polymer towards the template during the loading step. The
high reloadability of extracted MI systems is another
peculiar characteristic of the system. It was reported
in literature [106] that MI contact lenses were applied
in vivo for the ocular release of timolol showing the
significant enhancement of properties in respect to the
not imprinted control in terms of drug loading (1.6fold higher after extraction of the template and reloading) and consequently the released drug dose (23-fold
higher). MI systems with increased drug rebinding
affinity, in contact lenses (for the release of timolol[107]
and other drugs [108112] or wetting agents[113]), in
transdermal systems [114,115] , have been extensively
reported in the literature.
It has been demonstrated that multiple noncovalent
site of interactions can delay the release of diclofenac
sodium and ethyl adenine-9-acetate from acrylic MI

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materials, composed of diethylaminoethyl methacrylate and 2-hydroxyethylmethacrylate [116,117] , opening the way to zero-order kinetics release systems.
The literature reports the use of these materials for
the delivery of chemotherapic agents, such as 5-fluorouracil [118] , metal-based drugs [119] , anti-inflammatory [120,121] , anticonvulsant [122] , proton pump
inhibitor [123] and for the enantioselective release of
therapeutic agents[123125] . This interesting MI application consists of a controlled release of one enantiomer of a racemic mixture of active principle (Figure 4) .
The necessity to release only one of the enantiomers
is related to the different pharmacological activity
that drugs can exert: the favorable release of the more
active enantiomer promotes a better therapeutic effect.
Racemic mixtures are cheaper to produce than pure
single enantiomers and also separation of enantiomers
after production is not straightforward in many cases.
Another interesting result obtained by combining
drug delivery systems with MI technology is the possi-

Racemic mixture

Imprinting solution

Review

bility to obtain a kinetics that is responsive in the presence of a specific molecule. This is the case in the study
reported by Sreenevasar [126] . This work reports an MI
system composed of 2-hydroxy ethyl methacrylate and
EGDMA and imprinted against hydrocortisone. The
system was capable of releasing the drug of interest
(testosterone) with a significantly enhanced kinetics in
the presence of the template molecule.
The synthetic approach in the preparation of the MI
systems can influence the delivery kinetics and the final
properties of the polymers. A systematic study of recognition properties combined to delivery performances
were evaluated in the work of Salian et al. [127] . In this
paper the synergic effect of the synthesis scheme (living radical polymerization vs conventional free radical
polymerization) and MI technology was demonstrated.
Obtained gels showed an increased template binding
capacity and decreased delivery kinetics parameters.
Attempts to further control the delivery kinetics
were made by using environmental stimuli-responsive

MIP

Extracted MIP

Drug release over time

Drug-loaded MIPs
(specific + non-specific absorption)

Released drug

Reloading mixture

Time
Figure 4. Molecular imprinting particles toward enantiomers. Particles are imprinted toward one of the enantiomers
of the racemate; after the template removal, obtained MIPs can be used for two different applications:
(A) separation of racemic mixtures; and (B) enantioselective-controlled delivery of racemic drugs.
MIP: Molecular imprinting particle.

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MI materials. The review of Puoci et al. [128] summarizes the recent literature on the stimuli-responsive MI
polymers.
Drug delivery & molecularly imprinted
carriers: a potential winning strategy
The use of polymeric nanoparticles as drug carriers
is currently one of the most challenging topics in
nanomedicine. Thanks to their properties nanocarriers can be directly injected into the bloodstream
allowing fast and efficient systemic treatment. Their
ability to protect the drug from the reticuloendothelial system allows a lower dose to be administered,
with fewer side effects for the patient. However, the
preparation and optimization of nanoparticles for
the administration of drugs are not straightforward.
The optimization of MI nanoparticles may take
several aspects into account. In addition to the customary properties of conventional drug nanocarriers
(e.g., biocompatibility, hemocompatibility), specific
properties related to the MI structure (e.g., efficient
molecular recognition, high porosity) are requested.
Attempts to optimize at the same time all the functional properties of MI nanoparticles were done in
the work of Mirzaei etal. [129] . In this paper, particles imprinted against tetracycline and composed
of poly(methylmethacrilate) cross-linked with ethylenglycol dimethacrylate were synthesized and characterized. This study reported that the affinity of
particles towards the template molecule decreased by
increasing the porogen polarity, in addition particles
showed a significant selectivity for the template and a
sustained release in respect to nonimprinted particles.
Considering the prolonged delivery capability, MI
nanoparticles seem to be more interesting than conventional polymeric carriers. As such, efforts have
been made in the last years to develop these devices.
The prolonged pharmacological activity of MI particles may be useful if carriers are stable in the physiological environment for a suitable time. Conventional
nanoparticles have been widely in this respect, unlike
MI nanoparticles which still need to be improved by
combining the MI synthesis with appropriate surface
engineering strategies. The stability of nanoparticles
may be improved to prevent their aggregation. This
aspect was tackled in the work of Moczko et al. [130] .
They proposed a steric stabilization of nanoparticles by
attaching a poly(ethyleneglycol) corona on MI particle
surface, providing a higher stability in water, buffer
and blood protein solutions.
Molecular imprinting technology can be combined
with the specific physicochemical properties of the
used materials to obtain more complex and functional
systems.

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Apart from the smart recognition properties, MI

nanoparticles can be obtained as a gel-like system, as
reported in the work of Cirillo et al. [131] , where nanoparticles imprinted against 5-fluorouracile showed high
rebinding capacity and a sustained release thanks to their
hydrogel behavior. In addition to the control exerted by
the MI peculiar properties, the drug delivery kinetics
can be further tuned introducing a stimuli-responsive
behavior. A thermoresponsive N-isopropylacrylamidebased nanogel imprinted against lysozymes developed
by Pan et al. serves as an example of this [132] . NIPAAmbased materials are known in literature to show a behavior that is strongly dependent on the temperature. In
this work, MI nanoparticle showed a dramatic temperature-dependent behavior concerning rebinding kinetics,
swelling and release profiles. The major result obtained
was the significant delay of the protein release rate of MI
nanoparticles with the increase of the temperature in
respect to the behavior of nonimprinted nanoparticles
that did not show changes in the release kinetics with
the same parameter. Thus this work has demonstrated
that combining thermoresponsive monomers with the
MI technology it was possible to increase the control on
the drug delivery kinetics.
Passive targeting of MI particles can be obtained
by using external magnetic fields. In order to demonstrate this, a magnetic composite system for the controlled release of aspirin was reported in the work of
Kan etal. [133] . Polymeric nanoparticles composed of
methacrylic acid cross-linked with trimethylolpropane
trimethacrylate were synthesized in the presence of
magnetic iron oxide nanoparticles with the possibility
to prepare a system for the magnetic targeted delivery.
Future perspectives for molecular imprinting
nanoparticles
As previously summarized, molecular imprinting
technology can be exploited for the production of
stable and cheap materials, in the form of micro- or
nanoparticles, for in vivo sensing and diagnostic,
drug targeting and also as antidotes to bind and capture viruses, metals or toxins from the bloodstream.
The literature is constantly growing and some niche
applications have been already developed as starting point for the preparation of integrated and more
complex systems based on the use of MI polymers.
Interesting applications were proposed in recent years
and some studies are summarized in the following
paragraphs.
Synthetic antibodies to bind dangerous
antigens directly in the bloodstream

Antibodies are proteins able to selectively recognize

their target molecules on the basis of a combination

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Molecularly imprinted polymeric micro- & nano-particles

of interactions, like hydrogen bonding, electrostatic or

hydrophobic, with the three-dimensional structure of
the target. The similarity of MI polymers with these
biological molecules opened the way to the design of
completely synthetic antibodies composed of a polymeric material. Plastic antibodies can be obtained by
imprinting the polymer of interest toward a molecule,
a protein, a peptide or a fragment thereof. Although
the use of MI polymers for the adsorptive removal of
molecules in aqueous environment was widely proved,
the feasibility of the removal of molecules in the
bloodstream has not been clearly demonstrated. The
first attempts to produce synthetic antibodies were
made by Mosbach and co-workers [134] with application as radioimmunoassay. They prepared a molecularly imprinted system, composed of methacrylic acid
and ethylene dimethacrylate, for the evaluation of
drug levels (theophylline and diazepam) in human
serum through the inhibition of a radio-labelled ligand
binding by the serum analyte. Results were excellent
if compared with a well consolidated and commercial
technique, the enzyme multiplied immunoassay technique (EMIT), generally used for this assay. In a more
recent paper, Shea and co-workers [135] published an
interesting research in this field as important example
of biomimicry. They demonstrated that the injection
of acrylamide-based MI particles imprinted against
mellitin, a cytolytic peptide, showed significant results
in living mice in terms of increased survival rates
after the administration of the cytotoxic peptide and
MI nanoparticles. The recognition of the cytotoxic
substance and the consequent binding allowed the
elimination of the formed complex mellitin/nanoparticles by macrophages in the first thirty minutes after
the injection. This is the only one in vivo application
of MI synthetic antibodies available in the literature
and results seem to be very promising for further
developments.

Review

levels, foreseeing the possibility to control in situ the

balance between MMPs, retained by the scaffold,
and their inhibitors (TIMPs). Results allowed concluding that the balance MMPs/TIMPs can be regulated with the use of MI nanoparticles and without a
pharmacological therapy with the aim to prevent the
expansion of post-infarct left ventricular remodeling.
The reported study was focused on the retention of
MMP-9 but the concept of an enzyme-specific scaffold for tissue engineering may be extended also to
other molecules.
One of the most fascinating molecular
imprinting particles outlooks: the Trojan horses

When a pharmacological therapy is based on cytotoxic (e.g., chemotherapeutic agents) or hydrophilic

drugs, the internalization of the active principle
within cells can be difficult to achieve and the pharmacological therapy can fail because of the extremely
low drug uptake that lead to an ineffective intracellular level of the active principle. In these cases, the
efficacy of the treatment can be enhanced by using
a Trojan horse. The concept of Trojan horse is currently defined in literature in different ways. However, authors are in agreement with Collett et al. [137]
that have defined the Trojan horses as strategies that

MIP
MMP
TIMP
Myocardium (post infarct), high MMPs/TIMPs ratio

Regulation of the level of a specific solute in

the blood: a drug-free medical therapy

Considering the capability of MI polymers to bind

specific molecules, their use to impoverish a pathological site in a host body can lead to the regulation
of some balances of specific biomolecules in the tissues. This is the case of the study reported in the
paper of Cristallini and co-workers [136] (Figure 5) .
In this paper a biodegradable scaffold mimicking the
geometry of the extracellular matrix of the cardiac
tissue was loaded with MI nanoparticles. Particles
were imprinted against the matrix metalloproteinase
9 (MMP-9), an enzyme involved in wound repair
of cardiac tissue after infarction. The proposed system was evaluated for the in vitro control of MMP-9

future science group

Figure 5. Advanced applications of molecular imprinting

particles, regulation of the blood level of specific
molecules. MIPs can capture and retain the molecule in
excess restoring a correct physiological balance.
MIP: Molecular imprinting particles; MMP: Matrix
metalloproteinase; TMP: Tissue inhibitors of
metalloproteinase.

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Review Gagliardi& Mazzolai

combine, in the same engineered entity, a targeting
unit and a specific drug/gene delivery system. More
practically and focusing only on nanoparticles, Trojan horses are a particular class of carriers, composed
of a shell with high cell internalization capability and
a core containing the drug to be administered. The
shell with the Trojan horse behavior is able to hide the
active principle to the physiological repulsive response
of cell membranes, to bind a specific site and, consequently, to be internalized with high efficiency into
cells together with the hidden drug via conventional
endocytocis pathways.
The therapeutic approach based on the use of
Trojan horse nanoparticles was reported in literature only for conventional not imprinted nanoparticles. As an example, a Trojan horse can be obtained
by chemical conjugation on the particle surface of
glycans [138] , cell-penetrating peptides [139] or by
incorporating particles into monocytes [140] . A possible strategy to simplify the setup of a Trojan carrier could be the application of the MI technology.
Surface imprinting against a membrane receptor or
a fragment thereof can be a way to obtain receptorbinding particles that can be more easily internalized via a receptor-mediated pathway (Figure 6) . In
addition, another strategy could be to imprint the
nanoparticles against a small molecule that is abundant in the pathological site and internalized only by
diseased cells. The use of the MI technology instead
A

Cell to be treated,
overexpressed receptors

Cell to be treated

Figure 6. Advanced applications of molecular

imprinting particles, the Trojan horse. (A) The drug
carrier imprinted against a receptor or a fragment
thereof is internalized by receptor-mediated
endocytosis; (B) molecular imprinting particles are
imprinted toward a molecule that is abundant at the
pathological site and simply uptaken by the cells.

132

Future Med. Chem. (2015) 7(2)

of the common chemical conjugation or the use of

biological materials (peptides, proteins or cells) can
significantly simplify the design and the preparation
of the Trojan carrier and improve the stability and
the reliability of the system.
Molecular imprinting particles of the future:
to implement recognition properties on
biodegradable polymers

As previously mentioned, the interest for molecularly

imprinted drug delivery systems has grown over the
last decade. Developed systems that had previously
been described were composed of stable polymers. This
means that, after administration, particles can remain
in the bloodstream or in the tissue causing a bioaccumulation that is dangerous for cells and organs. Thus,
in order to increase the biocompatibility, biodegradable
MI nanocarriers can be evaluated.
Biodegradable polymers can be cleaved and more
easily eliminated by the body. Degradation products
are generally well tolerated and for these reasons biodegradable materials are interesting in biomedical
materials research. In the case of MI polymers, the
use of biodegradable materials is really limited because
of the intrinsic characteristic of these materials to
be degraded and to lose their original shape, with a
progressive decreasing of the recognition properties.
However, some attempts were made by the group
of Kim and co-workers [141143] . The Kim research
group reported three biodegradable systems, based on
poly(-caprolactone), poly(3-hydroxybutyrate) and
poly(lactic acid), end-functionalized with acrylic or
methacrylic groups, obtained in the form of membranes
after photopolymerization together with other acrylic
monomers. The template molecule used was theophylline and the selectivity of membranes was evaluated
against the template molecule and the homologous
caffeine. The selectivity of obtained membranes was
higher than for the nonimprinted polymer (3, 2.3 and
1.6times), indicating that the MI technology can also
work with biodegradable materials. Functional properties of the biodegradable materials were not affected
by the crosslinking. The biodegradation kinetics was
reported only for the first two systems, resulting in
10% and 15% weight loss after 30days. These values are comparable to those obtained for the respective
non crosslinked polymers.
Conclusion
Molecular imprinting is a versatile chemical strategy to obtain synthetic molecule-specific structures
in the form of monoliths, membranes, gels and
particles. The synthesis of MI polymers enables to
introduce functionalized cavities into the formed

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Molecularly imprinted polymeric micro- & nano-particles

polymer that provide molecular recognition capabilities. Functional cavities can recognize the template
molecule on the basis of the size, the shape but also
the topochemistry. In addition to the recognition
properties, molecularly imprinted cavities can retain
the molecule used during the imprinting process,
opening the possibility to obtain molecule-selective
separation systems. Molecular imprinting is a cheap
process but the optimization of the final products
may require significant efforts. MI polymers show
interesting properties that can be exploited in the
biomedical field and in nanomedicine. Sensors, synthetic immunoassays, molecular separation devices
or scaffolds for tissue engineering can be produced
by using customized MI polymers with great advantages in terms of stability, sensitivity, reduced costs
and reproducibility. Molecular recognition properties on MI polymers may completely substitute biologically-derived molecules in biohybrid polymeric
systems (e.g. peptide-functionalized particles), with
a significant increase of the safety of final products.
MIPs can find potential uses in nanomedicine as
excipients or drug carriers. The application of MI in
drug delivery systems preparation is still at an early
stage but promising proof of concepts have been
developed and well documented in the literature.
There are several advantaged in the exploitation of
MIPs as drug carrier, mainly related to the delivery
kinetics. MI polymers can significantly prolong the
delivery, foreseeing the possibility of a zero-order

Review

kinetics. Furthermore, obtained systems are reloadable providing the possibility to re-trigger in vivo
an exhaust system after the complete release of the
starting drug cargo. Completely synthetic targeted
drug delivery systems can be prepared via molecular
imprinting technique with an opportune choice of
the template molecule. Thus, drug-loaded surfaceimprinted particles against proteins, nutrients or
receptors involved in physiological cell internalization processes may show high internalization capabilities. Moreover, MIPs can be used as themselves
for a drug-free treatment involving the removal, from
the blood or a pathological site, of metals, viruses,
bacteria, enzymes or other different molecules causing a disease. The constantly growing literature on
molecularly imprinted polymers confirms the great
potential of this technology, opening the way to their
actual application as drug delivery systems, scaffold for tissue engineering, membranes for specific
separations, sensors and immunoassays.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment,
consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this
manuscript.

Executive summary
Molecular imprinting (MI) is a chemical procedure that is used to introduce molecular recognition properties
in polymers in the form of monoliths, membranes, gels and particles.
The imprinted polymer is obtained around a target molecule (template) that is removed after the synthesis,
leaving molecular cavities complementary in size, shape and chemical functionalities to the extracted
template.
Molecular recognition properties of MI polymers are enough specific toward the template molecule that they
provide the recognition of the template in respect to homologous and enantiomers.
Synthetic immunoassays based on MI polymers are more stable and cheaper than biological ones.
Molecular sensors composed of MI polymers are more sensitive than conventional ones.
MI membranes are useful for molecular separation based on the chemical properties of the molecule to be
retained and not on its molecular dimension; this property opens a way to more efficient dialysis processes.
MI scaffolds represent a further enhancement of micro- or nanopatterned scaffolds for tissue engineering
providing a more controlled guidance of cell growth.
Drug delivery based on MI particles results more controlled in terms of release kinetics, providing more
prolonged administrations than conventional systems and showing the capability to be reloaded after the
complete release of the drug cargo.
MIPs can immobilize and remove pathogens from the bloodstream (synthetic antibodies) or specific molecules
in a pathological site to restore the physiological conditions.
A targeted drug delivery system can be obtained from particles imprinted against cell receptors or other
molecules abundant at the pathological site (Trojan horse).
The development of biodegradable MI polymers represents a big challenge and it allows combining high
biocompatibility with molecular recognition properties.

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Review Gagliardi& Mazzolai

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Future Med. Chem. (2015) 7(2)

This letter reports one of the most important works

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Very interesting in vivo research that reports encouraging

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