Targeted drug delivery by aptamer-modified nanoparticles

Drug delivery systems are systems able to release a drug in a controlled way, so that can
modulate the release speed, the dose, the residence time, and, above all, discriminate the
site where the presence of the active principle is required. The methods currently used to
administer the drug have drug-kinetic restrictions due to the lack of simultaneity between
the time necessary for the value of the drug concentration to be adequate for the therapy,
and the trend of the medicine release due to diffusion; in this way, the drug is distributed at
the systemic level, and the effectiveness of the drug is achieved only when the
concentration at the site of action reaches a certain value, which is obtained by
administering high and repeated doses over time. This leads to the formation of rather toxic
secondary effects. Consequently, the conventional therapies are not a convenient response
to the needs for an adequate treatment of serious diseases such as cancer. Especially in
recent decades, the interest has moved towards the search for alternative solutions aimed at
minimizing the side effects and at the identification of highly compatible materials able to
selectively recognize the site of action, releasing the drug on site [1, 2].
An approach that is certainly attracting particular interest in the pharmaceutical and
biotechnological field, is represented by the so-called "targeted drug delivery", a method
by which there is the possibility of selectively and quantitatively transporting a drug
directly to the site where it has to perform its action.
The targeted drug delivery can be conducted by intervening on the design and
implementation of three fundamental elements, which, combined together, allow to obtain
a highly efficient product, as it acts selectively, with greater intensity and each carrier is
associated with more drug molecules. These components are represented by the drug, the
targeting molecule and the nanocarrier that can be made up of colloidal structures such as
liposomes, metallic nanoparticles, polymers, nanogels, micelles and dendrimers, as can be
seen in Figure 1A. For example, polymeric nanogels are highly biocompatible, and exhibit
sensitivity to changes in pH, temperature or ionic strength, which gives the system the
programmed and "intelligent" release capacity at the site of action; for this reason they
have been studied with great attention for several decades as potential solutions to
problems in the biomedical field. In fact the properties of specific microenvironments can
be exploited to allow the nanosystem to carry out a controlled release, or by means of an
external treatment mediated by ultrasounds or electromagnetic fields [3]. Another example
can be given by the change of structure of some polymeric nanocarriers that, in particular
microenvironments, cause the release of the drug [4] and it has been seen that this
condition can be widely exploited in the treatment of tumors, because neoplastic cells have
an altered metabolism affecting the pH of the tissue and the redox potential [5]. In fact it
has been shown that in many types of tumors, in particular the solid ones, an increase in
the concentration of glutathione in the cytosol is found; this particularity can be exploited
for the design of functionalized nanoparticles through the use of thiol groups in order to
allow controlled release in a reducing environment [6].
The nanosystems must be modified on the surface, so that they can assume some important
characteristics for their functioning, such as biocompatibility, and the possibility to
selectively bind to particular receptors, for active directioning. The concept of targeted
drug delivery is based on the selectivity of the nanocarriers used to transport the drug in a
specific site of action and on the appropriate target that must be chosen based on its
accessibility characteristics, but also on the drug that must be incorporated and conveyed.
Actually, the directioning of nano-systems to the site of action can take place by means of
two modalities which act in a passive or active way.
Passive directioning is a process that depends on the chemical-physical characteristics of
the nanocarrier and on the physiological properties of the target site; the mechanism
underlying this process is the EPR (Enhanced Permeability Retention) effect, a
phenomenon described for the first time about 25 years ago, which consists of the
increased permeability and retention of molecules with appropriate dimensions that tend to
accumulate in the tumor tissue rather than healthy one. The phenomenon is due to the rapid
growth of the tumor mass that stimulates the formation of blood vessels with anomalous
shape and organization presenting fenestrations in the vascular endothelium, and the lack
of adequate lymphatic drainage; this entails a considerable reduction in the recovery of
macromolecules into the blood, as can be seen in Figure 1B.
This effect allows the accumulation of the drug incorporated into the vector, without the
aid of targeting molecules anchored on the surface increasing their selectivity towards the
target, but with the precaution of constructing a nanocarrier that is large enough to pass the
anomalous fenestrations [7, 8]. In spite of the interest that the EPR effect and its
potentialities arouse, the passive direction of a carrier towards the target site does not
always represent a great advantage, since a homogeneous and predictable distribution of
the drug in the neoplastic mass is not guaranteed. As a result, more and more often the
focus is on active targeting. This type of directioning is based on the high affinity
recognition between a specific ligand anchored on the surface of a nanocarrier and a
receptor overexpressed exclusively or predominantly on the target cells that constitute the
tissue to be treated, as can be seen in Figure 1C. Often the two modes of targeting act
independently and nanocarriers can be developed in order to operate either actively or
passively; in certain circumstances, however, these two modalities can be combined to
increase the specificity of the treatment to pathological tissues, trying to minimize the
harmful effects on healthy ones.

Figure 1. In (A) the fundamental elements of a drug delivery system are shown, while in (B) the image
highlights the EPR effect occuring in the areas of the organism affected by the tumor and how it can be
exploited for active and passive targeting (C).

Another very important concept for the development of a drug delivery system is the
clearance of the nanocarrier, since when these systems are present in the blood, they can be
recognized as non-self antigens, and be opsonized by adsorption of proteins such as
immunoglobulins, complement proteins or fibronectin on the surface of the particle, and
this facilitates the process by which the delivery system is recognized by monocytes and
phagocytes that phagocytate it in the liver [9]. This system represents a way of defense of
the organism, whose action depends on the dimensions and the superficial properties of the
nanoparticles, such as the presence of certain functional groups and the density of surface
charge, facilitating or not the interaction with the molecules of the blood, and for this
reason, it is necessary to take these properties into consideration when we want to design
these nanosystems [10].
The size of the nanocarriers has a fundamental importance, because this parameter
regulates the biodistribution and circulation of the nanoparticle, subjected to the hepatic
and renal action. Very small nanoparticles (smaller than 10 nm) are secreted in the kidneys,
whereas too large ones tend to be phagocytized by monocytes and reticulocytes.
The carrier-free systems based on aptamers as a means of targeting and delivery, rapidly
undergo renal clearance, due to their small size, so it was thought to combine the aptamers
with voluminous molecules such as PEG, cholesterol, proteins or nanoparticles of different
types so as to be able to provide those dimensional characteristics to the system, useful for
increasing the half-life in the organism, but also to improve the resistance of the system to
the degradation effected by the nucleases of the serum [11]. Another method used to
increase the size of the carrier-free delivery system was to bind together more aptamers in
order to have multimeric, but also multivalent complexes (in the case of use of different
aptamers), this method allowed an increase in the half-life in vivo [12].

Figure 2. Schematic illustration of the clearance of nanoparticles which highlights the importance of size and
zeta potential: the blue area of the graph represents the effects of the size, while the red area represents the
effects of the zeta potential.

In addiction, considering the width of the fenestrations present in the capillaries that
irrigate the tumor tissue, it has been seen that the useful dimensions to pass therethrough
are approximate to 10-100nm, sizes that also allow the nanoparticle to remain in the
bloodstream for more time [13]. The importance of the size of these systems is also evident
in the role that they play in activating complement proteins, because, below 200 nm in
diameter, the nanoparticles do not seem to show activity; whereas, overcome these
quantities, they are quickly eliminated in the liver. As for the superficial properties of the
nanoparticles, the charge density plays an important role in the interaction with the blood
proteins and with the surface of the cells present in the bloodstream, and can cause
aggregation of the nanoparticles in a physiological environment (this phenomenon has
been observed in particular in polymeric dendrimers and in quantum dots). In addition to
size, surface properties also play a key role in the clearance of nanocarriers and it has been
seen that, if they are covered by appropriate polymers, they can become invisible and
evade the body's defenses, whereas if their surface is too hydrophilic, the nanoparticles can
be drained and undergo rapid clearance [14]; the effects of size and zeta potential are
shown in Figure 2.
Another important process to consider for the design of a delivery nanosystem is the
administration of the nanocarrier, by which the drug reaches the site of action and
according to which the elimination of the xenobiotic can occur in different ways.
Depending on the route of administration, the medicine may have a local or systemic
effect; in the first case, the administration consists of injecting the drug directly into the
part to be treated or in its proximity, and in this way it does not enter the circulatory
system. Systemic administration, on the other hand, occurs when the drug enters the
bloodstream and reaches the site of action, which is not necessarily close to the area of
application. The main systemic pathways are parenteral pathways, through mucous
membranes, epithelia or by intravenous, intra-arterial, intradermal, subcutaneous or
intramuscular injection, and enteral pathways, which are the oral, rectal and sublingual
routes. If the administration is done by injection into the blood stream, the clearance of the
particles occurs by elimination in the spleen and in the liver, operated by macrophages,
whereas if the injection is the interstitial one, the nanoparticles reach the nearby lymph
nodes, passing through the endothelial cells of the capillaries that present adhesions that
are not strictly adherent, since these organs are dedicated to the drainage of the lymph.
Then, the particles reach the lymphatic node and, according to their size, they can be
phagocytosed by macrophages (in the case of particles larger than 100 nm) or be drained
from the blood capillaries [15]. These considerations are at the basis for the design of the
type of drug delivery system, which will also depend on the type of pathology, the
physiological and anatomical conditions, and the specific recognition between nanocarriers
and target cells. Remaining in the topic of active targeting, the ligands used to make the
system specific to the target, belong to the class of antibodies, peptides, carbohydrates, or
aptamers. The aptamers are proving very advantageous, as they can recognize and bind a
wide variety of molecules with high affinity and specificity and they also have lower
immunogenicity and size than the other ligands, thus allowing better tissue penetration. As
a consequence, the high specificity of recognition of aptamers can be widely exploited for
the administration of drugs, chemotherapeutic molecules in particular, since xenobiotics
often have poor selectivity for target cells and non-selective treatment causes the onset of
side effects also serious on healthy cells. For example, antibiotics such as anthracyclines
(daunorubicin or doxorubicin) are used for the treatment of various tumors, but often cause
high toxicity because, as they are intercalators, they can be nonspecifically intercalated in
the double stranded DNA or RNA cytosine-guanine sequences. On the other hand, one of
the problems associated with aptamers is their instability at the hematic level; in fact the
half-life of the aptamers is about 10 minutes, since they are easily degraded by nucleases
[16]. In order to make aptamers more stable in this compartment, a way to overcome this
drawback is the modification of the their molecular structure, and this can occur through
different strategies including sugar modifications, 3' end and phosphodiester bond
modifications, as shown in Figure 3. As for the sugar modification, substitution with an
amine, O-methyl or fluorine group in position 2’ can be obtained and it has been shown
that this strategy makes the pyrimidines (cytosine, thymine or uracil) less susceptible to the
nuclease action (especially ribonuclease). In other cases, the aptamers can be modified by
inserting analogs in which the sugar is modified with an extra bridge connecting the 2'-O
and 4'-C, obtaining the LNA (locked nucleic acid), which greatly increases the resistance
to the nuclease degradation. Other analogues have also been developed in order to increase
the folding, for example the UNA (unlocked nucleic acid), without C2'-C3 binding [17,
18]. As for  the 3' end, it is chemically modified to resist nucleases, such as the 3'-
exonuclease, for example by binding an inverted thymidine to it; this modification has
been used to obtain therapeutic aptamers such as Pegaptanib and other nucleic acid-based
drugs [19]. Another modification of the 3' end is the bind with an inverted biotin; Dougan
et al. also used streptavidin to modify the thrombin aptamer, resulting in a 3'-biotin-
streptavidin binding and they showed that this modification increased the resistance to
nucleases [20]. In order to obtain more nuclease resistant aptamers, it is also possible to
modify the phosphodiester bond and replace it, for example with methylphosphonate,
phosphorodiozoate or sulphonate, or converting the D nucleic acids into their enantiomeric
form L (called Spiegelmers) [21].

Figure 3. Schematic representation of modifications on aptamers, in order to make them more resistant to
nucleases and to increase their half-life.

The main problem arising as a result of the modification of aptamers is the reduction of
affinity with their ligands caused by structural change; consequently, the most important
study should be directed to the final aptamer, analyzing how and with how much intensity
it binds to its target after its modification [22]. Recently, some research groups have
developed new polymerases able to use modified analogues, as precursors for the synthesis
of aptamers, and these enzymes can be used during the SELEX process. In this way, the
synthesis and selection process of the aptamer with a higher affinity lead to the formation
of a product already modified, in order to be potentially more resistant to nucleases,
without the need for further steps that could compromise the final structure and
consequently the affinity. For example, aptamers with nucleotide analogs in their structure
can be synthesized, using the RNA polymerase of a T6 mutant, or nucleotide modifications
may occur using KOD Dash DNA polymerase in the PCR cycles [23, 24]. Recently, the
rapid development of nanomaterials has allowed these systems to be included in drug
delivery studies.
Nanomaterials are defined as materials having a size of less than 100 nm. The
characteristics that allow to change the properties of nanomaterials are essentially the
chemical composition (changes in the physical-chemical behavior), the size (solubility
changes, absorption or emission wavelength, conductivity, melting point) and the surface
(changes in dispersion capacity, conductivity, and through functionalization with different
molecole and more appropriate ligands it is possible to have a decrease in aggregation and
bond selectivity). Hence their choice is due to the fact that they have these advantageous
physical-chemical properties. Among the most used nanomaterials as carriers, liposomes,
metallic and/or magnetic nanoparticles, Quantum Dot, albumin nanoparticles, polymeric
nanoparticles and dendrimers are considered. The aptamers can be conjugated to different
nanomaterials, for example the dendrimers, for the targeted drug delivery and to date, some
hundreds of aptamers have been isolated and their affinity has been determined with a
series of biological targets, such as antigens of the surface of cell membranes and growth
factors [25]. Another important aspect is the loading strategy of the drug into the system. It
can be carried out in different ways and the most used are: doping, by means of which
drugs can be incorporated into liposomes, micelles or silica nanoparticles, and conjugation
due to non-covalent bonds, electrostatic attraction or covalent bond, which allows the
therapeutic molecule to be linked to the carrier.

Aptamer-conjugated nanomaterials.

Aptamer-conjugated liposomes.
One of the nanomaterials to be taken into high consideration is the liposome, since this
carrier has very useful features such as an aqueous internal environment and hydrophobic
portion of the membrane, which allow to encapsulate both hydrophobic and hydrophilic
drugs, as showm in Figure 4. Moreover, in order to prolong the residence time of the
liposome in the blood and consequently to obtain a more efficient accumulation at the site
of action, various molecules, such as polyethylene glycol, can be bound on the surface of
the particle; in particular, molecules such as PEG increase the biocompatibility of the
delivery system, an important aspect if we want to obtain an innovative therapy that is less
toxic to the body. As a result, liposomes have been extensively studied for drug delivery
and they are well prepared to be used as selective carriers, combining them with aptamers
to increase recognition and targeting efficiency on cells to be treated. It is necessary to
underline that the liposomes present a therapeutic efficacy and reduce the side effects of
many drugs even in the absence of aptamer, and this has been shown by encapsulating
Doxorubicin in PEGylated liposomes (obtaining Doxil product, approved by the FDA for
clinical use [26]), but active targeting with the use of receptors, such as aptamers, allows a
further improvement in the effectiveness of the drug. An early design of this delivery
sistem was developerd in 2009 by Cao et al.; they synthesized a PEGylated liposome and
subsequently immobilized the anti-nucleolin AS1411 aptamer on its surface through the
use of a spacer molecule functionalized with cholesterol in order to anchore it to the
phospholipid bilayer. To provide the carrier with antitumour activity and to monitor it,
cisplatin (with antiproliferative action) and a hydrophilic dye were respectively
encapsulated in the internal aqueous environment. The following cell viability assays
confirmed that this system could specifically release cisplatin into cancer cells [27]. Xing
et al. used the same system, consisting of the AS1411 aptamer immobilized on the
liposome by means of cholesterol bound on the nucleic acid and they incorporated
doxorubicin into the internal aqueous environment of the nanoparticle; they then tested the
delivery system on breast cancer cells and found that, compared to receptor-free liposomes,
the liposomes conjugated with AS1411 were more toxic. The complex was then injected
into mice with mammary carcinoma and in this case it was seen that the liposome
conjugated with the aptamer had a greater antitumor activity too. [28]. In subsequent works
Xing et al. investigated the effects of the spacer, varying its length and chemical nature,
and noting that these characteristics were important for targeting efficacy of the aptamer
[29]. Some studies developed a system in which the aptamer was modified with FITC
fluorophore and it was bound on the surface of PEGylated liposomes using the reaction
between thiol, present on the nucleic acid, and maleimide present on the free end of PEG.
In this way Alshaer et al. used an aptamper able to bind to the CD44 receptor, a protein
overexpressed by many tumor cells, demonstrating that the conjugate had significantly
greater binding affinity than the free-aptamer liposome [30]. Subsequently, this aptamer-
liposome complex was called "aptamosome" by Baek et al. and in 2014 they developed a
delivery system consisting of PEGylated liposome conjugated with an aptamer able to bind
to the prostate-specific membrane antigen (PSMA) through the post-insertion method:
micelles previously conjugated with the aptamer were prepared and they were then
incubated with PEGylated liposomes loaded with Doxorubicin, in order to obtain
aptamosomes. Once the delivery system was produced, the complex was tested on cell
cultures and on mice in which prostate cancer was xenografted; it has been shown that the
aptamosomes were significantly more toxic to the cancer cells in vitro, and, administered
intravenously to the rodents, they exhibited antitumor activity showed by the decrease in
tumor mass in vivo [31]. Recently, another potential cure for prostate cancer cells by in
vivo administration has been developed by Stuart et al.; they designed a liposomal complex
conjugated with anti-PSMA aptamer and inside it TPEN (N, N, N ', N'-Tetrakis (2-
Pyridylmethyl) -Ethendiamine) was encapsulated, a zinc chelator that decreases the
intracellular level of zinc and induces apoptosis. Even in this case the greater antitumor
efficacy of the aptamosome has been demonstrated, compared to the simple drug-loaded
liposome, reducing the mass of prostate human cancer xenografted in rodents [32].

Figure 4. Schematic representation of a liposome-based delivery system; it is possible to notice the different
types of drugs that can be transported and the different ways of binding the aptamer on its surface, by means
cholesterol or PEG.
Aptamer-conjugated nanoparticles.
Other nanomaterials used as carriers are gold nanoparticles, as they possess excellent
characteristics, such as biocompatibility, stability, low toxicity and ease of modification
because of the large surface that can be functionalized with aptamers by means of the Au-
thiol group bond. Furthermore, the gold nanoparticles can have dimensions such as to be
subjected to EPR effect and accumulate in the tumor tissues. However, unlike liposome-
based systems, which have an accessible internal environment, the nanoparticle structure
functionalized with aptamer may limit the number and types of drugs that can be
transported; principally these systems carry chemotherapies, and, since most of them are
intercalators, they can be inserted between the nucleic acid bases, as shown in Figure 5.
In recent years, several studies have been carried out for the design, modification and
improvement of aptamer-AuNP systems for cancer therapy; for example, Luo et al.
developed a DNA-sg8c aptamer based delivery system specific for the protein tyrosine
kinase 7 (PTK7), a truncated receptor upregulated in many common human tumors, and a
hairpin DNA bound on the AuNPs surface. The chemotherapeutic doxorubicin was then
intercalated into the repeated sequence present in the hairpin structure. The complex was
tested to demonstrate its antitumor efficacy and low toxicity, and it was seen that when it
was illuminated with an appropriate light beam (in the resonance plasmonic wavelength),
the complex showed a greater antitumor activity [33].
Hollow gold nanospheres (HAuNS) can also be used as a carrier for the delivery system
and an additional feature of these complexes is the quick release of the drug, controlled by
various factors such as pH change. For example, Zhao et al. designed HAuNS in which
specific RNA aptamers for CD30 (biomarker used to diagnose Hodgkin's lymphoma) and
with doxorubicin intercalated between the bases, were covalently bound to the metal
surface. Furthermore, this system has proved to be stable at physiological pH (7.4), but
under conditions of acidic environment (5.0) the complex became ultrasensitive, releasing
most of doxorubicin (a situation that can occur in the lysosomal environment). Therefore,
by means of the characteristics of ultrasensitivity to pH and of specific recognition due to
the functionalization with aptamer, the system quickly and effectively released the
chemotherapic in lymphoma cells, but didn't shown a relevant toxicity to CD30 negative
control cells [34].
Another work demonstrating a pH-controlled drug release was performed by Danesh et al.
who used AuNPs functionalized with anti-PTK7 aptamer and loaded with the intercalator
daunorubicin (a chemotherapic drug used for the treatment of lymphoblastic leukemia,
whose administration in patients can cause several side effects, such as cardiotoxicity).
Moreover, it was verified that these complexes effectively released the drug in an acid
environment and, following toxicity studies conducted on target and control cells, their
cytotoxicity was assessed, demonstrating greater specificity and intensity than free drug or
intercalated daunorubicin in the bases of free aptamer [35].
The therapeutic efficacy of the drug-loaded AuNP-Aptamer complex can be further
improved by using two types of targeting molecules, in what is called a dual targeting of
the delivery system. For example, the first targeting molecule can bind to an extracellular
receptor, whereas the second one can bind to a cytoplasmic or nuclear protein. In
particular, the administration of these nanoparticles may prove useful in reducing side
effects related to the treatment, as the multi-functionality of these systems allows the
release of the drug to target cells in an even more specific way. Chen et al. designed a
complex consisting of a gold nanocluster conjugated with the cRGD peptide, an αvβ3
integrin ligand (overexpressed in tumor cells) and an aptamer binding nucleoline (also
overexpressed in the cytoplasm and nucleus of tumor cells). The system was further
functionalized with a fluorescent dye (NIR) for imaging or alternatively loaded with the
chemotherapeutic doxorubicin for therapy and evaluation of antitumor efficacy. From the
analysis carried out, they have seen that the complex has been efficiently internalized in
cancer cells in vitro (mainly in the nucleus, but also in the cytoplasm) and it has
accumulated in the tumor tissue in vivo (by administering it on mice to which glioma was
xenografted), carrying out an inhibiting activity on the growth of the cancerous mass.
Moreover, no serious toxicity was found in healthy tissues [36].
Another variant of the AuNP-based system conjugated with aptamers involves the release
of several therapeutic molecules and it is called multidrug delivery. This approach is
proving to be an important strategy especially to improve the efficacy of anticancer
therapies, as one of the major problems leading to the failure of chemotherapies is the
development of drug resistance by cancer cells. A combined and simultaneous action by
multiple drugs in therapy may allow a greater success rate of healing. Shiao et al.
developed a release system consisting of AuNPs conjugated with the AS1411 aptamer, to
which a photosensitive porphyrin (TMPyP4, tetra-(N-methyl-4-pyridyl) porphyrin) and
doxorubicin were non-covalently attached. The complex was then tested on target cells,
resistant doxorubicin cells and control cells; the results showed that exposure to light
induced the production of reactive oxygen species (carried out by TMPyP4) inside the
cells, and in combination with the release of doxorubicin, the toxicity towards the target
cells was greater than in a single treatment, highlighting a synergy of therapeutic effects
between the two drugs [37].
Lastly, in addition to AuNPs, other types of nanomaterials can be used in a drug delivery
system, such as carbon nanotubes, gold nanorods and graphene-related materials, and the
conjugation of the drug in the nanoparticle-aptamer complex not only occur by means of
intercalation in the bases of the nucleic acid, but it can also occur by loading on the surface
of the nanomaterial through hydrophobic interactions or covalent binding.

Figure 5. Schematic illustration of an AuNPs-based delivery system; it is possible to notice different types of
drugs that can be transported, and the different ways of binding the aptamer on its surface, by means a linker
or a capture complementary DNA.

Aptamer-conjugated polymeric micelles and polymersomes.

The choice of nanomaterials for use as a carrier is not only limited on the liposomes or
AuNPs, but involves polymeric micelles and polymersomes, as shown in Figure 6. The
micelles are composed of lipids or amphiphilic molecules, which self-assemble forming
vesicles with a hydrophobic core which allows to transport lipophilic drugs. They have
been studied as carrier since 1984 [38] and their use has proved to be very interesting for
the characteristics of biocompatibility, biodegradability and for the possibility of
functionalization with targeting molecules. The micelles are obtained from copolymers
consisting of two or more types of monomer units, which differ in their polarity and length:
generally, when the hydrophilic segment is longer than the hydrophobic one the micelles
take on a spherical shape, whereas, if the length of the hydrophobic portion increases with
respect to the hydrophilic segment, structures with rod and reed morphology can be
formed. Micelles are conjugated with aptamers to get active targeting and can be of
different types; for example Dhar et al. used poly (D, L-lactic-co-glycolic) nanoparticles
(PLGA) as carrier for the administration of cisplatin, demonstrating a selective therapeutic
efficacy for tumor cells and a residence time in the blood in in vivo studies [39, 40].
Polymersomes are a class of artificial vesicles consisting of amphiphilic copolymers,
which internally have an aqueous solution used to encapsulate drugs of a hydrophilic
nature; instead, the inside of the hydrophobic membrane may be the place inside which
hydrophobic drugs are trapped. Their surface can be functionalized with aptamers, for
example Alibolandi et al. developed a system consisting of PEG-PLGA nanopolymers
loaded with doxorubicin and functionalised with EpCAM aptamer on the surface.
Subsequently, the conjugate was tested on MCF-7 cells and was shown to be much more
toxic on target cells than the ligand-free conjugate [41].

Figure 6. Schematic illustration of a delivery system based on micelles (on the left) and polymersomes (on
the right); drugs with different chemical characteristics can be transported.

Aptamer-conjugated dendrimers.
Dendrimers are synthetic polymers consisting of highly regular and branched structures
with a polyfunctional core surrounded by repeated units, and surface functional groups that
confer characteristics to these nanomaterials. Their synthesis takes place by means of
repeated condensations of the same branched unit, by reacting a diamine with methyl
acrylate, and starting from the core representing the nucleation center, from which
ancestral branches dipart. From these ones, other ramifications branch off forming a
structure called dendrone and each subsequent branching takes the name of generation.
The drugs used in the therapy are usually loaded into the central portion of the dendrimer
because it has a greater porosity than the peripheral portion and their release depends on
the properties of the chains. Furthermore, by means of terminal groups, it is possible to
functionalize the surface of the nanoparticle with aptamers for the active targeting, and
PEG, in order to confer stability and "stealth" properties to macrophages, as shown in
Figure 7. A class of dendrimers that has been found useful for the release of drugs and
biomolecules in various cell types, is made from polyamidoamines (PAMAM), since they
have a higher biocompatibility due to their internal structure consisting of amide bonds,
similar to the structure of proteins globular. For example, Lee et al. developed a release
system consisting of PAMAM dendrimers functionalized on their surface with single-
stranded oligodeoxynucleotides, hybridized with anti-PSMA aptamers. Subsequently, they
loaded the conjugate with doxorubicin, intercalating it in the double strand and they tested
it in a model of prostate tumor in vivo, showing specificity and antitumor properties [42].
Drug delivery can also take place through the functionalization of several types of
aptamers on the surface of the dendrimers. For example, Taghdisi et al. developed a
conjugate consisting of dendrimers modified with three aptamers (binding MUC1,
nucleolin and ATP) for the targeted release of epirubicin. The system was then tested in
vitro on C26 cells (mouse colon carcinoma cells) and in vivo, injecting it into mice with
colon carcinoma, demonstrating targeted targeting efficacy specific for target cells in the
first case and inhibition of tumor growth in the second case [43].

Figure 7. Schematic representation of a dendrimer-based delivery system; the loading location of the drug
(internal, superficial or intercalation) is highlighted.

Carrier-free delivery.
Over the years, drug delivery systems that do not require the use of nanoparticles have
been developed, although the aptamer continues to be present. In these cases, in fact the
ligand is conjugated directly to the therapeutic molecule through covalent or non-covalent
binding for active targeting. For example, the conjugation of drugs, in particular
chemotherapeutics, with aptamers binding over-expressed receptors on the membrane of
tumor cells, has proved useful for reducing the side effects of the drug occuring at a
systemic level. One of these drugs, doxorubicin, was used as a model for aptamer-drug
conjugation studies and carrier-free targeted delivery and it was chosen the intercalation
into the nucleic acid bases as the preferential loading method. Bagalkot et al. developed a
carrier-free release system by combining doxorubicin with PSMA binding aptamer and
tested its efficacy on LNCaP (human prostatic adenocarcinoma cells) cultures; the obtained
results showed a greater antitumor activity compared to different cell cultures lacking
PSMA [44]. Alternatively, doxorubicin can be covalently bound to the aptamer by the
formation of amide bonds, disulfide or esters; this method was introduced because simple
intercalation in nucleic acids may present the disadvantage of uncontrolled release of the
drug before reaching the site of action. Huang et al. developed this method, combining the
drug with sgc8c DNA aptamer by means of hydrazine as a linker, and this allowed to
increase the antitumor activity on CEM (acute lymphoblastic leukemia) cells [45]. In
addition to doxorubicin, other drugs can be conjugated; for example, Zhao et al. covalently
conjugated methotrexate with a DNA aptamer binding CD117 receptor, over-expressed on
AML cells. The obtained results confirmed the specificity and the increased
chemotherapeutic activity [46].
Since the post-transcriptional regulation mechanism based on siRNA and miRNA has been
discovered, in the following years it was decided to use these oligonucleotides as
therapeutic molecules, in the modulation of the expression of oncogenes for the treatment
of tumor pathologies. For example has been possible to conjugate them with aptamers for
their controlled and effective release within cells, and siRNAs linked to aptamers are the
most used oligonucleotides. These conjugates represent a good system of siRNA release in
tumor cells, in particular epithelial and stem cells. In a recent work, Subramanian et al.
developed a release system using an aptamer binding EpCAM (membrane-associated,
tumor-associated antigen) conjugated to a specific siRNA to PLK1 (Polo-like kinase 1);
after having tested the complex on MCF-7, they showed a strong antitumor activity, with
regression of the cancerous mass [47]. Recently, using the same EpCAM aptamer, Wang et
al. succeeded in reducing the gene expression of survivin (an inhibitory protein of the
caspase, involved in the apoptotic pathway) by combining the corresponding siRNA with
the nucleic acid; testing the complex in mice with xenografted cancer, they showed a
regression of doxorubicin chemioresistant cancer stem cells [48].

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