**3. Aptamers**

In the 1980s molecular virology revealed that small structured oligonucleotides could bind proteins with high affinity and specificity. That evidence supported the use of oligonucleoti‐ des as specific receptors, which 10 years later lead to the discovery of aptamers [58]. The word "aptamer" was first used in 1990 by Ellington and Szostak to describe small RNAs molecules able to bind small organic dyes. It derives from the fusion of the Latin expression "aptus", which means "to fit", and the Greek word "meros", which means "part" [59]. Since then, aptamers have been defined as short oligonucleotides that by adopting specific 3D conformations are able to bind specific and selected targets [60].

Aptamers are mostly short single-stranded or double-stranded DNA or RNA oligonucleoti‐ des, usually 20–80 bp long and 6–30 kDa heavy. Aptamers are constituted of a random sequence region at center, flanked by constant designed primer binding sites and the 3′ and 5′ ends. The sequence region in the center is necessary for target recognition (**Figure 1**), which occurs after aptamer 3D adaptation. In this phenomenon intermolecular interactions, such as Van der Waals forces, hydrogen and electrostatic interactions, stabilize the bond between aptamers and their ligands [61, 62]. The aptamers-ligands interactions are highly specific and capable to discriminate among analogues, i.e., enantioselective aptamers have 12.000-fold higher affinity for L-arginine than for D-arginine [63].

Aptamers are thought to be an excellent alternative to the use of monoclonal antibodies (mAB). Compared with antibodies, aptamers overcome their issues and improve their clinical applicability and suitability for industrialization. First of all, aptamers are low-immunogenic and low-toxic molecules, and they are not directly recognized by the human immune system as foreign agents [64–66]. Unlike antibodies, aptamers have a wider target range, they are smaller so that they can easily penetrate into tissue barriers and cells [67]; moreover they can also bind small ligands, such as ions and small molecules, which cannot be recognized by antibodies [68]. Furthermore, aptamers are thermally stable, and can undergo repeated cycles of denaturation/renaturation without damaging their binding efficiency. Finally, aptamer production and eventually modification is cheaper, easier and faster than that of mAB [68].

The interest of research on aptamers is increasing, as shown by the publication rate on this topic, which has exponentially grown in 25 years [61], leading to more than 5500 published articles in the PubMed database including the term "aptamer" in January 2016. In spite of their popularity, their clinical applications are still limited [62], and as of today only one aptamerbased drug has been approved by the US Food and Drug Administration (FDA). Pfizer/Eyetech launched Macugen, a RNA aptamer against VEGF (vascular endothelial growth factor) for the treatment of wet age-related macular degeneration in 2004 [69]. Barriers to the commerciali‐ zation of aptamers are essentially two. The former is that some *in vitro* generated aptamers do not elicit a comparable *in vivo* comparable, whereas the latteris that the SELEX process is timeconsuming and not very efficient [62]. In spite of these issues, a recent market report project‐ ed the global aptamer market to \$5.4 billion by 2019 [70].

**Figure 1.** Diagram representing aptamer 3D conformational rearrangement in the presence of the target to form aptam‐ er-target specific complex.

#### **3.1. Aptamers generation**

All the issues connected to the strategy described, prompted us to develop a new method to enhance scaffold biocompatibility by using a novel class of molecules, called aptamers, to

In the 1980s molecular virology revealed that small structured oligonucleotides could bind proteins with high affinity and specificity. That evidence supported the use of oligonucleoti‐ des as specific receptors, which 10 years later lead to the discovery of aptamers [58]. The word "aptamer" was first used in 1990 by Ellington and Szostak to describe small RNAs molecules able to bind small organic dyes. It derives from the fusion of the Latin expression "aptus", which means "to fit", and the Greek word "meros", which means "part" [59]. Since then, aptamers have been defined as short oligonucleotides that by adopting specific 3D

Aptamers are mostly short single-stranded or double-stranded DNA or RNA oligonucleoti‐ des, usually 20–80 bp long and 6–30 kDa heavy. Aptamers are constituted of a random sequence region at center, flanked by constant designed primer binding sites and the 3′ and 5′ ends. The sequence region in the center is necessary for target recognition (**Figure 1**), which occurs after aptamer 3D adaptation. In this phenomenon intermolecular interactions, such as Van der Waals forces, hydrogen and electrostatic interactions, stabilize the bond between aptamers and their ligands [61, 62]. The aptamers-ligands interactions are highly specific and capable to discriminate among analogues, i.e., enantioselective aptamers have 12.000-fold

Aptamers are thought to be an excellent alternative to the use of monoclonal antibodies (mAB). Compared with antibodies, aptamers overcome their issues and improve their clinical applicability and suitability for industrialization. First of all, aptamers are low-immunogenic and low-toxic molecules, and they are not directly recognized by the human immune system as foreign agents [64–66]. Unlike antibodies, aptamers have a wider target range, they are smaller so that they can easily penetrate into tissue barriers and cells [67]; moreover they can also bind small ligands, such as ions and small molecules, which cannot be recognized by antibodies [68]. Furthermore, aptamers are thermally stable, and can undergo repeated cycles of denaturation/renaturation without damaging their binding efficiency. Finally, aptamer production and eventually modification is cheaper, easier and faster than that of mAB [68]. The interest of research on aptamers is increasing, as shown by the publication rate on this topic, which has exponentially grown in 25 years [61], leading to more than 5500 published articles in the PubMed database including the term "aptamer" in January 2016. In spite of their popularity, their clinical applications are still limited [62], and as of today only one aptamerbased drug has been approved by the US Food and Drug Administration (FDA). Pfizer/Eyetech launched Macugen, a RNA aptamer against VEGF (vascular endothelial growth factor) for the treatment of wet age-related macular degeneration in 2004 [69]. Barriers to the commerciali‐ zation of aptamers are essentially two. The former is that some *in vitro* generated aptamers do

improve protein adsorption and cell adhesion.

334 Advanced Techniques in Bone Regeneration

conformations are able to bind specific and selected targets [60].

higher affinity for L-arginine than for D-arginine [63].

**3. Aptamers**

Aptamer selection requires two steps: upstream screening and downstream screening. The upstream screening step identifies full-length aptamers through SELEX (Systematic Evolu‐ tion of Ligands by EXponential Enrichment), whereas the downstream step aims to isolate the shortest oligonucleotide sequence required for target binding [61].

#### *3.1.1. Upstream screening*

*In vitro* selection or SELEX (Systematic Evolutions of Ligands by EXponential enrichment) is the technique used to isolate aptamers, which was first described by Ellington and Gold in 1990 [59, 71].

**Figure 2.** Schematic representation of the SELEX process steps.

The SELEX process consists of three steps, which are then repeated to screen for sequences with higher affinity (**Figure 2**) [58]: (a) the preparation of an initial oligonucleotides pool (library) is followed by (b) the selection of aptamer candidates and by (c) their amplification.

#### *3.1.1.1. Library generation*

The whole SELEX process starts with the generation of a synthetic oligonucleotides library, which consists of a pool of ~1012 –1015 different nucleic acid (ssDNA or RNA) sequences, theoretically able to bind any target molecule. Each sequence represents a possible aptamer candidate and it possesses a central random region, ~25–30 bp long, flanked by two stand‐ ard primers at the 3′ and 5′ ends [61, 62].

Both ssDNA and RNA libraries can be created and divided in five types, on the basis of the collected sequences. Standard libraries are the most common ones and contain random 20–60 bp long oligonucleotides. Structurally constrained libraries contain oligonucleotides with stable regions, which help aptamers to fold according to a certain secondary structure. Libraries based on a known sequence are constructed by inserting known sequences in the central part of the oligonucleotide. Finally, libraries based on genomic sequences (genomic SELEX) are created by digesting genomic DNA, to find proteins capable to bind it [72].

#### *3.1.1.2. Binding and separation*

Once the library is generated, it is incubated with the target. Some of the oligonucleotides in the pool recognize the target and are then considered aptamers (partitioning), whereas unbound sequences are filtered out from the solution (elution) [61].

Several methods are used to discriminate aptamers from other oligonucleotides. The SELEX approach initially employed by Gold and co-workers was based on a nitrocellulose mem‐ brane where the T4 DNA polymerase was immobilized [71]. Nowadays, the use of a nitrocel‐ lulose membrane is quite out of order because it has some limitations, such as the inability to bind any molecules but proteins and the need to perform at least 12 selection rounds [73, 74]. Alternative strategies have then been developed based on common biochemistry techniques. Chromatographic affinity or magnetic columns are often used for aptamer selection. In the case of chromatographic affinity column, the immobile phase is composed of agarose beads and the targets are immobilized through tags with proteins, such as glutathione *S*-transfer‐ ase (GST) or His-tag, or through chemical reaction with other molecules. Several aptamers have been selected through this method, however it cannot be applied if the target lacks the tags or the functional group requested for the coupling with the column [75–77]. On the other hand, targets can be immobilized on the surface of magnetic beads and used in magnetic columns, a strategy that is becoming more and more powerful due to the ease of separating aptamers from other nucleotides only by a magnet [78–80]. Furthermore, capillary electro‐ phoresis has been proposed, because of its speed and high resolution. In fact a successful selection of aptamers can be obtained in a few rounds, i.e., Bowser and co-workers selected aptamer against neuropeptide Y and against human IgE in only four rounds [81, 82]. In addition to the methods described above, aptamers against whole cells have been recently selected through the Cell-SELEX method. This technique is complex, because cells cannot be immobilized, unlike target molecules; however, several research groups have been success‐ ful. Kobatake's group identified the SBC3, a cell lung cancer cell line with a ssDNA aptam‐ ers [83]. Previously, Tan's group selected a series of aptamers able to bind two types of ovarian cancer cells [84], whereas Gold et al. isolated an aptamer for the U251 cell line derived from glioblastomas just in 2003 [85].

Further strategies have been implemented to improve SELEX performance, although their efficiency in selecting aptamers is not still clear.

#### *3.1.1.3. Amplification*

The SELEX process consists of three steps, which are then repeated to screen for sequences with higher affinity (**Figure 2**) [58]: (a) the preparation of an initial oligonucleotides pool (library) is followed by (b) the selection of aptamer candidates and by (c) their amplification.

The whole SELEX process starts with the generation of a synthetic oligonucleotides library, which consists of a pool of ~1012 –1015 different nucleic acid (ssDNA or RNA) sequences, theoretically able to bind any target molecule. Each sequence represents a possible aptamer candidate and it possesses a central random region, ~25–30 bp long, flanked by two stand‐

Both ssDNA and RNA libraries can be created and divided in five types, on the basis of the collected sequences. Standard libraries are the most common ones and contain random 20–60 bp long oligonucleotides. Structurally constrained libraries contain oligonucleotides with stable regions, which help aptamers to fold according to a certain secondary structure. Libraries based on a known sequence are constructed by inserting known sequences in the central part of the oligonucleotide. Finally, libraries based on genomic sequences (genomic SELEX) are created by digesting genomic DNA, to find proteins capable to bind it [72].

Once the library is generated, it is incubated with the target. Some of the oligonucleotides in the pool recognize the target and are then considered aptamers (partitioning), whereas

Several methods are used to discriminate aptamers from other oligonucleotides. The SELEX approach initially employed by Gold and co-workers was based on a nitrocellulose mem‐ brane where the T4 DNA polymerase was immobilized [71]. Nowadays, the use of a nitrocel‐ lulose membrane is quite out of order because it has some limitations, such as the inability to bind any molecules but proteins and the need to perform at least 12 selection rounds [73, 74]. Alternative strategies have then been developed based on common biochemistry techniques. Chromatographic affinity or magnetic columns are often used for aptamer selection. In the case of chromatographic affinity column, the immobile phase is composed of agarose beads and the targets are immobilized through tags with proteins, such as glutathione *S*-transfer‐ ase (GST) or His-tag, or through chemical reaction with other molecules. Several aptamers have been selected through this method, however it cannot be applied if the target lacks the tags or the functional group requested for the coupling with the column [75–77]. On the other hand, targets can be immobilized on the surface of magnetic beads and used in magnetic columns, a strategy that is becoming more and more powerful due to the ease of separating aptamers from other nucleotides only by a magnet [78–80]. Furthermore, capillary electro‐ phoresis has been proposed, because of its speed and high resolution. In fact a successful selection of aptamers can be obtained in a few rounds, i.e., Bowser and co-workers selected aptamer against neuropeptide Y and against human IgE in only four rounds [81, 82]. In addition to the methods described above, aptamers against whole cells have been recently

unbound sequences are filtered out from the solution (elution) [61].

*3.1.1.1. Library generation*

336 Advanced Techniques in Bone Regeneration

*3.1.1.2. Binding and separation*

ard primers at the 3′ and 5′ ends [61, 62].

After the separation of aptamers from a specific nucleotides, they are amplified by PCR, in the case of ssDNA aptamers, or by RT-PCR in the case of RNA aptamers. Consequently, prod‐ ucts of amplification are used as a new sub-library for the following selection round [62].

#### *3.1.2. Downstream screening*

After the upstream screening or SELEX, selected aptamers are generally ~80 bp long, but the binding region is actually usually 10–15 nucleotides long [68, 86]. As a consequence, redun‐ dant and useless nucleotides can be deleted through a process called "aptamer truncation". Many strategies have been tested to minimize aptamer sequences without damaging its binding ability. Most of these strategies are predictive and based on computational biology. Giangrande et al. were able to truncate an RNA aptamer against PSMA (prostate-specific membrane antigen) while preserving its binding activity and functionality, using structure simulations and target docking algorithms. Partial fragmentation was used by Green et al. to select the shortest sequence of DNA aptamers to bind PDGF (platelet-derived growth factor) [87]. Wang and co-workers detected the non-essentialregion ofthe hPTK7 (anti-human protein tyrosine kinase 7), hybridized them with complementary oligonucleotides probes [88]; the same approach was used by Duan and co-workers to select the basic region of anti-CD133 aptamer as marker for cancer stem cells [89].

All the methods described for aptamer truncation are effective; however, their application is hindered by their complexity, length, and cost [61].

#### **3.2. Biomedical applications of aptamers**

The similarities between aptamers and mABs lead to their applications in various field, including research tools [90], bioassays [91, 92], food safety [93], and environment monitor‐ ing [94], as demonstrated by a plethora of reviews recently published on this topic. However, a major field of interest for aptamers is biomedicine, where aptamers can be used as sensors for biomarker discovery, molecular imaging probes, drug delivery systems and drugs, especially in cancer nanomedicine and therapy [58, 61].

#### *3.2.1. Aptamers as potential drugs*

Although the most studied aptamers are against thrombin, VEGF and PDGF, aptamer applications range from cancer to infectious pathogens.

#### *3.2.1.1. Therapeutic aptamers in eye disease*

The first therapeutic aptamer approved by the FDA was the Pegaptanib, which today is commercially available as Macugen® (Pfizer and Eyetech) [64, 65]. The Pegaptanib is a 27 ribonucleotide pegylated RNA aptamer antagonist of VEGF165 [95]. Since its approval in 2004, the Macugen® has always been used for the treatment of AMD, a degenerative ocular disease that causes vision loss in older adults due to retinal damage. However, the efficacy of this aptamer was then discovered to be important also for the treatment of diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) with promising results in clinical trials [96, 97]. At present, the spectrum of use of Pegaptanib is being broadened to other pathologies such as ischemic diabetic macular edema (MIDME), uveitis, choroidal neovascu‐ larization secondary to pathologic myopia, and iris neovascularization [98–100].

The limits of anti-VEGF agents to treat AMD are their inability to promote the regression of new blood vessels, which are the cause for the loss of vision. To bypass this limitation, the E10030 aptamer (Fovista™) was developed by Ophthotech Corp in 2012: the E10030 is a 29 pegylated RNA aptamer able to bind PDGF (platelet-derived growth factor), which regu‐ lates pericytes maturation. The combined administration of E10030 with Pegaptanib showed successful neovascular regression in preclinical models [101].

#### *3.2.1.2. Therapeutic aptamers for hemostasis*

Thrombin is a wide-studied target for anticoagulation, and its *in vivo* inhibition is a major solution to prevent and treat blood clotting abnormalities [61, 102]. Anti-thrombin aptamer (TBA), a 15 bp oligonucleotide, was first selected in 1992 by Toole et al. and it was the most studied aptamer for clinical applications in 2012 [60, 103]. After the evaluation of TBA efficiency *in vivo* [104], the Nu172 aptamer (ARCA Bipharma) was develop as a potential thrombin inhibitor candidate. Nu172 is a 26 bp aptamer able to prevent fibrinogen cleavage of a-thrombin by interacting with the exosite I. Nu172 is currently in phase II clinical trials to be certified for anticoagulation in invasive medical procedures, coronary artery bypass graft and percutaneous interventions [105].

#### *3.2.1.3. Therapeutic aptamers for cancer*

The goal of new therapeutic approaches in Oncology is often to block the neoplastic progres‐ sion through the inhibition of specific cell-pathways, which lead to cell abnormal prolifera‐ tion. Several clinical trials have proposed the use of aptamers to specifically bind tumor cells and stop cancer development. The specific cell membrane receptors that can be blocked in tumors are numerous, but only few have been investigated with aptamers. A pivot role is played by nucleolin, a protein which is often over-expressed on the surface of cancer cells and that is firstly involved in cell survival, growth and proliferation, as well as in nuclear trans‐ port and transcription [106]. In particular, nucleolin seems to manage the internalization of the tumor-homing F3 peptide and its inhibition affects several signaling pathways responsible for abnormal cell proliferation during cancer progression, such as NF-kB and Bcl-2 pathways [107, 108].

AS1411 (Antisoma, PLC) is a 26 bp long aptamer rich in guanosine and screened for against nucleolin [66, 106]. When AS1411 interacts with surface nucleolin, the complex is internal‐ ized and prevents its binding with Bcl-2, thus inducing cell apoptosis. AS1411 has shown good growth-inhibitory properties *in vitro* (**Table 1**) and the ability to be accumulated in tumor tissue [66, 109].


**Table 1.** Dose administered and time of exposure of different cell lines to AS1411 aptamer, in order to observe growth ihnibition [66, 109].

#### *3.2.1.4. Therapeutic aptamers in microbiology*

*3.2.1. Aptamers as potential drugs*

338 Advanced Techniques in Bone Regeneration

*3.2.1.1. Therapeutic aptamers in eye disease*

applications range from cancer to infectious pathogens.

Although the most studied aptamers are against thrombin, VEGF and PDGF, aptamer

The first therapeutic aptamer approved by the FDA was the Pegaptanib, which today is commercially available as Macugen® (Pfizer and Eyetech) [64, 65]. The Pegaptanib is a 27 ribonucleotide pegylated RNA aptamer antagonist of VEGF165 [95]. Since its approval in 2004, the Macugen® has always been used for the treatment of AMD, a degenerative ocular disease that causes vision loss in older adults due to retinal damage. However, the efficacy of this aptamer was then discovered to be important also for the treatment of diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) with promising results in clinical trials [96, 97]. At present, the spectrum of use of Pegaptanib is being broadened to other pathologies such as ischemic diabetic macular edema (MIDME), uveitis, choroidal neovascu‐

The limits of anti-VEGF agents to treat AMD are their inability to promote the regression of new blood vessels, which are the cause for the loss of vision. To bypass this limitation, the E10030 aptamer (Fovista™) was developed by Ophthotech Corp in 2012: the E10030 is a 29 pegylated RNA aptamer able to bind PDGF (platelet-derived growth factor), which regu‐ lates pericytes maturation. The combined administration of E10030 with Pegaptanib showed

Thrombin is a wide-studied target for anticoagulation, and its *in vivo* inhibition is a major solution to prevent and treat blood clotting abnormalities [61, 102]. Anti-thrombin aptamer (TBA), a 15 bp oligonucleotide, was first selected in 1992 by Toole et al. and it was the most studied aptamer for clinical applications in 2012 [60, 103]. After the evaluation of TBA efficiency *in vivo* [104], the Nu172 aptamer (ARCA Bipharma) was develop as a potential thrombin inhibitor candidate. Nu172 is a 26 bp aptamer able to prevent fibrinogen cleavage of a-thrombin by interacting with the exosite I. Nu172 is currently in phase II clinical trials to be certified for anticoagulation in invasive medical procedures, coronary artery bypass graft

The goal of new therapeutic approaches in Oncology is often to block the neoplastic progres‐ sion through the inhibition of specific cell-pathways, which lead to cell abnormal prolifera‐ tion. Several clinical trials have proposed the use of aptamers to specifically bind tumor cells and stop cancer development. The specific cell membrane receptors that can be blocked in tumors are numerous, but only few have been investigated with aptamers. A pivot role is played by nucleolin, a protein which is often over-expressed on the surface of cancer cells and that is firstly involved in cell survival, growth and proliferation, as well as in nuclear trans‐

larization secondary to pathologic myopia, and iris neovascularization [98–100].

successful neovascular regression in preclinical models [101].

*3.2.1.2. Therapeutic aptamers for hemostasis*

and percutaneous interventions [105].

*3.2.1.3. Therapeutic aptamers for cancer*

When aptamers were first described by Ellington and Gold in 1990, their ability to bind viral proteins was clear, and, consequently, their use to treat viral and bacterial diseases has always been investigated [110, 111].

Ebola epidemic of 2014 and other emerging viruses have prompted several research groups to use specific aptamers in the treatment of these diseases by blocking sites essential for virus infectious progression [112–115]. For example, it has been shown that specific aptamers against influenza major targets are able to inhibit or block virus fusion, penetration, and replication [116–120]. Aptamers are also thought to be useful to kill multidrug-resistant (MDR) bacteria *in vivo*, possibly by blocking resistance enzymes such as NMD-1 (New Delhi metallo-βlactamase) or by inducing the classic pathway of the complement in lieu of antibodies [121– 124].

#### *3.2.2. Aptamers as sensors for biomarker discovery*

Biomarkers are molecules that change their expression level when physiological conditions are altered, and can thus be used to indicate the progression state of a disease or the risk of

developing it. Biomarkers are therefore a tool with high potentiality for disease screening and early diagnosis. However, a very limited number of biomarkers have been thus far discov‐ ered. The use of mABs to identify disease specific targets is often unfeasible, because these targets are frequently cell epitopes and it is impossible to design and select a mAB against an unknown receptor, and aptamer research is moving to fill the gap. Normally, target cells are amplified, collected, and lysed. The lysate is then incubated with aptamers and target proteins go through a comparative proteomic analysis: briefly, they are separated through the SDS-PAGE and analyzed with mass spectrometry [61].

In recent years, many research groups have worked to find aptamers that specifically bind biomarkers. In particular, the tyrosine kinase 7 has been discovered as a potent marker of Tcell acute lymphoblastic leukemia [125], tenascin-C as biomarker for glioblastoma cells [85], the Igμ heavy chain for Burkitt's lymphoma [126], whereas the stress-induced phosphopro‐ tein I for ovarian cancer [127].

### *3.2.3. Aptamers as molecular imaging probes for diagnostic*

Aptamers have also been proposed as detecting agents in diagnostics, both as molecular beacons or as sensors for bio-imaging [58, 61].

In 2001, Hamaguchi et al. developed an aptamer beacon for thrombin. A thrombin aptamer was modified with complementary sequences at 3′ and 5′ ends to form a stem-loop struc‐ ture. Furthermore, the 5′-end was labeled with a fluorescent moiety, whereas the 3′ with a quencher. In the absence of thrombin, the complementary 3′ and 5′ ends lie in close proximi‐ ty and this results in fluorescence quenching, whereas in the presence of thrombin, aptamer acquires its 3D specific conformation, moving the fluorophore and the quencher apart, setting off a fluorescence signal in a dose-depends manner [128]. One year later the same approach was proved by Tan and co-workers [129] and then by several research groups [79, 130–132].

The idea of labeling aptamers with fluorophores was pursued also to develop new probes for computerize tomography (CT) and for magnetic resonance imaging (MRI). In addition, this idea seemed appealing in combination with nanomaterials for CT and MRI analysis (i.e., liposomes, quantum dots (QDs), carbon nanotubes, gold and magnetic nanoparticles), to improve *in vivo* imaging and phototermal therapy, thanks to aptamers' accurate targeting and the rapid diffusion through blood circulation of nanomaterials [58]. This approach was used to image C6 cancer cells using a Cy3-labeled aptamer against nucleolin transmembrane protein in 2010 [133]. The same year Min et al. proposed the use of a QDs-aptamer complex specific for PSMA(+) and PSMA(–) (prostate specific membrane antigen) to detect prostate cancer cells. The complex was able to discriminate between prostate cancer cells and normal or other cancer cells [134]. In 2013, Kim et al. immobilized a VEGFR2 (vascular endothelial growth factor receptor 2) aptamer on a magnetic nanocrystal surface for the detection of the angiogenic vasculature in glioblastoma by MRI with high sensitivity and efficiency [135].

#### *3.2.4. Aptamers as drug delivery systems*

developing it. Biomarkers are therefore a tool with high potentiality for disease screening and early diagnosis. However, a very limited number of biomarkers have been thus far discov‐ ered. The use of mABs to identify disease specific targets is often unfeasible, because these targets are frequently cell epitopes and it is impossible to design and select a mAB against an unknown receptor, and aptamer research is moving to fill the gap. Normally, target cells are amplified, collected, and lysed. The lysate is then incubated with aptamers and target proteins go through a comparative proteomic analysis: briefly, they are separated through the SDS-

In recent years, many research groups have worked to find aptamers that specifically bind biomarkers. In particular, the tyrosine kinase 7 has been discovered as a potent marker of Tcell acute lymphoblastic leukemia [125], tenascin-C as biomarker for glioblastoma cells [85], the Igμ heavy chain for Burkitt's lymphoma [126], whereas the stress-induced phosphopro‐

Aptamers have also been proposed as detecting agents in diagnostics, both as molecular

In 2001, Hamaguchi et al. developed an aptamer beacon for thrombin. A thrombin aptamer was modified with complementary sequences at 3′ and 5′ ends to form a stem-loop struc‐ ture. Furthermore, the 5′-end was labeled with a fluorescent moiety, whereas the 3′ with a quencher. In the absence of thrombin, the complementary 3′ and 5′ ends lie in close proximi‐ ty and this results in fluorescence quenching, whereas in the presence of thrombin, aptamer acquires its 3D specific conformation, moving the fluorophore and the quencher apart, setting off a fluorescence signal in a dose-depends manner [128]. One year later the same approach was proved by Tan and co-workers [129] and then by several research groups [79, 130–132].

The idea of labeling aptamers with fluorophores was pursued also to develop new probes for computerize tomography (CT) and for magnetic resonance imaging (MRI). In addition, this idea seemed appealing in combination with nanomaterials for CT and MRI analysis (i.e., liposomes, quantum dots (QDs), carbon nanotubes, gold and magnetic nanoparticles), to improve *in vivo* imaging and phototermal therapy, thanks to aptamers' accurate targeting and the rapid diffusion through blood circulation of nanomaterials [58]. This approach was used to image C6 cancer cells using a Cy3-labeled aptamer against nucleolin transmembrane protein in 2010 [133]. The same year Min et al. proposed the use of a QDs-aptamer complex specific for PSMA(+) and PSMA(–) (prostate specific membrane antigen) to detect prostate cancer cells. The complex was able to discriminate between prostate cancer cells and normal or other cancer cells [134]. In 2013, Kim et al. immobilized a VEGFR2 (vascular endothelial growth factor receptor 2) aptamer on a magnetic nanocrystal surface for the detection of the angiogenic

vasculature in glioblastoma by MRI with high sensitivity and efficiency [135].

PAGE and analyzed with mass spectrometry [61].

*3.2.3. Aptamers as molecular imaging probes for diagnostic*

beacons or as sensors for bio-imaging [58, 61].

tein I for ovarian cancer [127].

340 Advanced Techniques in Bone Regeneration

The ability of aptamers selected through the cell-SELEX to recognize cell antigens have been exploited to deliver a variety of molecules, mainly drugs, into cells [58]. For this purpose, aptamers can be used alone or in combination with other delivery systems, such as poly‐ mers or liposomes, in order to enhance their specificity [61].

Building on their previous work on a QDs-aptamer complex specific for PSMA(+) and PSMA(–) (see Section 3.2.3), Min et al. were able to load the construct with doxorubicin, an anticancer drug, and to effectively introduce it inside prostate cancer cells [136]. Levy's group relied on an aptamer against PSMA to introduce a siRNA in prostate cancer cells, which inhibited gene expression within 30 min [137].

To enhance polymers specificity as drug delivery system, they can be funtionalized with aptamers; this strategy has shown to be promising for clinical applications. Farokhzad et al. encapsulated rhodamine-labeled dextran within a nanoparticle of poly (lactic acid)-blockpolyethylene glycol copolymer with a terminal carboxylic acid functional group (PLA-PEG-COOH) conjugated to an aptamer against the PSMA antigen of prostate cancer cells. The system was able to enter PSMA over-expressing cells in less than 2 h [138]. The same group further generated a nanoparticle with poly (D,L-lactic-*co*-glycolic acid)-block-poly (ethylene glycol) (PLGA-*b*-PEG) copolymer conjugated with the A10 aptamer against PSMA to deliver docetaxel inside cancer cells. This system was tested *in vivo*, and induced the complete regression of the tumor in five out of seven mice [139]. Following these promising results, several others similar constructs based on the conjugation of polymers and aptamers were efficiently tested [140], and even aptamers conjugated to dendrimers were tested, as report‐ ed in a review published in 2011 by Lee et al. [141].

Liposomes were also engineered with aptamers to deliver cisplatin and taxol inside breast cancer cells. The AS1411 aptamer-liposome bioconjugate system efficiently transported cisplatin inside tumorigenic cells, and effectively killed the target cancer cells but not healthy control ones [142]. Moreover, compared with the control liposomes, liposomes conjugated with the AS1411 aptamer and containing taxol, increased the cellular uptake of the construct in the breast cancer cells [143].

Taken together, these results support the use of aptamers as enhancer for drug delivery systems; however, more *in vivo* evaluation is required to allow their use in clinic [61].
