The Use of Aptamers in Prostate Cancer A Systematic Review of

Clinical Biochemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem
Review
The use of aptamers in prostate cancer: A systematic review of

theranostic applications
Esther Campos-Fernández, Nathalia Oliveira Alqualo, Lorrayne Cristina Moura Garcia,
Caroline Coutinho Horácio Alves, Tábatha Danielle Ferreira Arantes Vieira,
Danilo Caixeta Moreira, Vivian Alonso-Goulart *
Laboratory of Nanobiotechnology, Institute of Biotechnology, Federal University of Uberlândia, Pará Avenue, 1720, Room 248, Building 2E, Umuarama Campus,
Uberlândia, MG 38405-320, Brazil
A R T I C L E I N F O A B S T R A C T
Keywords: Since prostate cancer (PCa) relies on limited diagnosis and therapies, more effective alternatives are needed.
Prostate cancer Aptamers are versatile tools that may be applied for better clinical management of PCa patients. This review
Aptamers shows the trends on aptamer-based applications for PCa to understand their future development. We searched
Theranostics
articles reporting aptamers applied in PCa on the Pubmed, Scopus and Web of Science databases over the last
Diagnostics
decade. Almost 80% of the articles used previously selected aptamers in novel approaches. However, cell-SELEX
Biosensors
Nanotechnology was the most applied technique for the selection of new aptamers allowing their binding to targets in their native
configuration. ssDNA aptamers were 24% more common than RNA aptamers. The most studied PCa-specific
aptamers were the DNA PSA-specific aptamer PSap4#5 and the PSMA-specific RNA aptamers A10 and A9,
being PSA and PSMA the most reported targets. Thus, researchers still prefer the ease of use of DNA aptamers.
Blood-based liquid biopsies represented 24% of all samples, being the most promising clinical samples. Especially
noteworthy, electro-analytical methods accounted for more than 40% of the diagnostic techniques and treatment
approaches with drug delivery systems or transcriptional modifiers were reported in 70% of the articles.
Although all these articles showed clinically relevant aptamers for PCa and there are good prospects for their use,
the development of all these strategies was in its early stages. Thus, the aptamers are not completely validated
and we foresee that the completion of clinical studies will allow the implementation of these aptamer-based
technologies in the clinical practice of PCa.
1. Introduction as much [3,4].

Currently, PCa diagnosis is made by measuring serum prostate spe
Prostate cancer (PCa) is the second most frequent malignancy after cific antigen (PSA), which is a serine protease also known as kallikrein-3
lung cancer among men worldwide. In 2018, 1,276,106 new cases of (KLK3) and secreted by cells in the prostatic epithelium in different
PCa and 358,989 deaths related to PCa were reported. This mortality situations such as PCa, hyperplasia, prostatitis, at advanced age, after
rate of PCa corresponds to 3.8% of all deaths caused by cancer in men in surgical interventions, ejaculation or hormone treatments [5]. PSA
that year [1]. measurement is applied in PCa screening and monitoring of this disease
Incidence and mortality of PCa across populations and over time progression, considering positive values for PCa the ones above 4.0 ng/
provide information about individual risk factors and population mL [6]. This cutoff usually leads to false positives that may bring about
screening [2]. Risk factors for PCa are age, family history, ethnicity and unnecessary biopsies, overdiagnosis and overtreatment [7].
inherited genetic factors. The incidence rates for African-American men Since PSA is not exclusive of the PCa condition, other biomarkers are
are higher when compared to Caucasian men, with 158.3 new cases being used in tests to determine whether a biopsy is necessary after
diagnosed per 100,000 men and their mortality is approximately twice finding high levels of PSA, helping to reduce unnecessary biopsies.
* Corresponding author.
E-mail addresses: [email protected] (E. Campos-Fernández), [email protected] (N. Oliveira Alqualo), [email protected] (L.C. Moura Garcia),
[email protected] (C. Coutinho Horácio Alves), [email protected] (T.D. Ferreira Arantes Vieira), [email protected] (D. Caixeta Moreira), alonso.
[email protected] (V. Alonso-Goulart).
https://doi.org/10.1016/j.clinbiochem.2021.03.014
Received 27 January 2021; Received in revised form 22 March 2021; Accepted 24 March 2021
Available online 29 March 2021
0009-9120/© 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Esther Campos-Fernández, Clinical Biochemistry, https://doi.org/10.1016/j.clinbiochem.2021.03.014
E. Campos-Fernández et al. Clinical Biochemistry xxx (xxxx) xxx
Prostate health index is a test that combines three forms of PSA: total 2. Materials and methods
PSA, free PSA and an inactive proenzyme form ([-2] pro-PSA). Similarly,
the 4 K score test measures four prostate-derived biomarkers: total PSA, 2.1. Literature search in PubMed, Scopus and Web of Science
free PSA, inactive PSA and human kallikrein 2. Both tests are performed
on blood samples. Other assays have been introduced into the clinical We performed a bibliographic search on three databases: PubMed,
practice, such as prostate cancer antigen 3 (PCA3), TMPRSS2-ERG, Scopus and Web of Science. The search in each database was made using
ExoDx and SelectMDx [8]. Some tests are applied after biopsy. For the same keywords and filters. The key words used for these searches
instance, Oncotype DX, which searches for 12 cancer-related genes; were “aptamer” AND “prostate cancer” AND (“prognosis” OR “diag
ProMark, which measures the expression of eight proteins for prognosis nosis” OR “treatment”). The searches were limited by the type of articles
and ConfirmMDx, which investigates DNA methylation. Few tests utilize that were journal articles (and clinical trials when available); the data,
circulating tumor cells (CTCs). The CellSearch system uses magnetic from 2010 to August 29th, 2019; and the language that was English. The
nanoparticles and antibodies to target epithelial adhesion molecules on articles obtained from these three databases were retrieved for further
CTCs for their enumeration and the Adna test analyzes CTC gene analysis.
expression by reverse transcription polymerase chain reaction (RT-PCR)
[7,9]. 2.2. Exclusion and inclusion criteria
Advances in our understanding of the epidemiology, diagnosis,
grading, staging, imaging and management of PCa have changed clinical Duplicate articles in the databases were excluded and then, titles and
practice and influenced guideline recommendations [10]. The hetero abstracts were analyzed to verify whether they met our filters for type of
geneous nature of PCa has encouraged more personalized treatment articles, data and language, and our eligibility criteria: articles published
strategies to boost positive clinical outcomes [9]. However, serum PSA in journals with an impact factor greater or equal to two, evaluating
and histopathological examination of tumor biopsies are still the gold aptamer performance and reporting aptamers specific for PCa bio
standards for PCa diagnostics and monitoring response to treatment. markers. Papers that did not meet the criteria were excluded and the
As the search for more specific biomarkers and more precise tech remaining ones were further assessed from their full texts to ensure that
niques that improve diagnosis, prognosis and prediction of treatments our criteria were met. The selected papers were read by two authors who
for PCa continues, targeted theranostic (diagnostic and therapeutic) filled in an excel table with data from the articles. Discrepancies be
agents with PCa-specific affinity molecules are promising approaches. tween these authors were solved by means of a discussion with a third
Monoclonal antibodies are widely available affinity molecules in the one.
healthcare sector. Nonetheless, chemical antibodies known as aptamers
show advantages compared to them. They both have a comparable af 2.3. Questions for the quality assessment of retrieved articles
finity in the nanomolar range, but aptamers show no immunogenicity,
being safer for clinical practice. Aptamers are more easily, quickly and The articles included from PubMed, Scopus and Web of Science da
economically produced due to their chemical synthesis and modifica tabases were analyzed according to their quality standards by nine
tion, which also yield no inter-batch variability. Aptamers are highly questions defined by the CASP Qualitative Checklist (questions 1, 2, 6, 7,
stable in extreme conditions, which guarantees a higher shelf life and 8 and 9) [17], the Quality Assessment of Diagnostic Accuracy Studies
easier storage and transport. Their nanometric size allows them to (QUADAS) (question 4) [18] and the Cochrane Handbook for Reviews of
interact with a wide variety of targets, from inorganic molecules to Diagnostic Test Accuracy (questions 5 and 9) [19]. The third question
whole cells and to penetrate tissues and internalize into cells. Further (3) was added by us to appraise the methodologies used in the articles.
more, they can be screened in the original biological conditions without For each retrieved article, the questions were answered by two authors
prior knowledge of the target molecule, thus allowing the discovery of who filled in an excel table with the answers “no”, “yes” or “unclear”.
unknown biomarkers [11]. Discrepancies between these authors were solved by means of a dis
Aptamers are short single-stranded DNA (ssDNA) or RNA molecules, cussion with a third one.
or short chains of amino acids that bind to target molecules with high The questions are listed as follows: 1. Was there a clear statement of
affinity thanks to their particular three-dimensional structures, which the aims of the research? 2. Was the research design appropriate to
allow them to form stable and specific complexes with their target address the aims of the research? 3. Did the study use appropriate and
molecules [12]. Thirty years ago, Tuerk and Gold published a paper relevant materials such as biological samples or in vivo models? 4. Was
where they selected the first nucleic acids by the Systematic Evolution of the execution of the index test described in sufficient detail to permit
Ligands by Exponential Enrichment (SELEX) technique [13]. Shortly replication of the test? 5. Did the study provide a clear definition of what
after, Ellington and Szostak also published a paper where they selected was considered to be a positive result? 6. If necessary, have ethical issues
nucleic acids by this technique and called them aptamers [14]. SELEX been taken into consideration? 7. Was the data analysis sufficiently
consists of sequential rounds of selection and amplification steps. An rigorous? 8. Is there a clear statement of findings? 9. Was the study free
initial synthetic oligonucleotide or peptide library of millions of random of commercial funding?
sequences is screened after iterative incubations with the target of in
terest in increasingly stringent conditions. Unbound sequences are 2.4. Data extraction
removed after every round resulting in the amplification of increasingly
specific binding molecules that are sequenced at the end of the process The following data were extracted from the articles that we included
[15]. Although aptamers have been known for three decades and in our analysis for their descriptive analysis and comparison in terms of
aptamer-based drugs have been evaluated in clinical trials, Pegaptanib is percentage: name and type of aptamers, aptamer refinements, origin of
the only aptamer commercialized until now. Macugen is its commercial aptamers, target biomarkers, clinical applications, samples and assays
name and its use is approved for the treatment of wet age-related used, and diagnostic techniques and type of treatments with their cor
macular degeneration in the United States, Europe, Canada, Brazil and responding administration route (Table 1).
Australia [16]. Therefore, there are some impediments that prevent
aptamers from reaching the market. The need for better clinical ap 3. Results and discussion
proaches for PCa makes aptamers very promising candidates as thera
nostic tools in this field. Thus, we aimed to identify trends in the use of 3.1. Retrieved articles
aptamers for the clinical practice of PCa to understand them and predict
future tendencies in this area. Initially, 258 articles were found. After eliminating duplicates and
2
Table 1
List of selected articles reporting aptamers for prostate cancer applications over the last decade.
Name of Refinements† Origin of Biomarker Clinical Biological samples and Diagnostic techniques Treatment
original aptamer applications assays approach
aptamer*
apt [75] – Chemical PSA Diagnosis Antigen in blood Fluorescence –

synthesis spectrometry
A9g [67] ●●● Chemical Exosomal PSMA Diagnosis Urine Fluorescence –
Nameless [76] ●● Chemical PSA Diagnosis Antigen in blood Impedance –
synthesis
iv
A10-3.2 [80] ●● Chemical PSMA Diagnosis Cell lines and mouse Ultrasound imaging –
synthesis xenograft model‡
AS2 and EpApt ● Chemical Exosomal PSA and Diagnosis and Cell lines and blood Colorimetry –
[78] synthesis EpCAM prognosis
PSap4#5 and ●●● Chemical PSA and VEGF Diagnosis Antigen in buffer Chemiluminometry –
V7t1 [45] synthesis
PSap4#5 [47] ●●● Chemical PSA Diagnosis Antigen in blood Voltammetry –
synthesis
E3 [20] ●●● Cell-SELEX LNCaP, 22Rv1, PC- Treatment Cell lines and mouse – Drugiv
3 and DU-145 cells xenograft model‡, Mfold‖
PSap4#5 [48] ●●● Chemical PSA Diagnosis Blood Impedance –
synthesis
PSap4#5 [49] ●●● Chemical PSA Diagnosis Blood Voltammetry and –
synthesis impedance
PSap4#5 and ●●●● Chemical PSA and VEGF Diagnosis Cell lines, Mfold
‖
Voltammetry –
V7t1 [50] synthesis
apt 1 [21] ●●● Cell-SELEX LNCaP cells Treatment Cell lines – Drug
PSap4#5 [51] ●●●● Chemical PSA Diagnosis Blood Voltammetry and –
synthesis impedance
A10-3.2 [59] ●●●●● Chemical PSMA Diagnosis Cell lines and mouse Ultrasound and –
synthesis xenograft model‡ fluorescence imagingiv
A10-3.2 [22] ●● Cell-SELEX PSMA Treatment Cell lines and mouse – Non-coding
xenograft model‡ RNAip
A10-3.2 [60] ● Chemical PSMA Diagnosis and Cell lines Photoacoustic imaging –
synthesis prognosis
AMH [31] ● Complementarity DBD of AR Treatment Cell lines, Gene Runner‖ – Direct
SOMAmers [29] ● Proprietary SELEX Exosomal protein Diagnosis Blood and urine Fluorescence –
array spectrometry
A10-3.2 [61] ●● Conventional Extracellular Treatment Cell lines and mouse – Non-coding
SELEX portion of PSMA xenograft model‡, RNAip
RNAstructure‖
A10-3-J1 [34] ●●●●● Chemical Extracellular Treatment Cell lines, Mfold‖ – Drug
synthesis portion of PSMA
IDA [23] – Cell-SELEX Integrin α6β4 Treatment Cell lines – Direct
PSap4#5 [52] ● Chemical PSA Diagnosis Antigen in blood Voltammetry –
synthesis
PSap4#5 [53] ● Chemical Free PSA Diagnosis and Antigen in buffer Chemiluminometry –
synthesis prognosis
A10-3 [62] ●●● Chemical PSMA Treatment Cell lines, mouse xenograft Non-coding
synthesis model and tissue sections‡ RNAiv
PSap4#5 [25] ●●●● Conventional PSA Diagnosis Blood and tissue sections Voltammetry and –
SELEX impedance
A10 [63] ●●●●●● Chemical Extracellular Treatment Cell lines, mouse xenograft – Drugiv
synthesis portion of PSMA model and tissue sections‡
PSap4#5 [35] ●● Chemical PSA Diagnosis Antigen in buffer Voltammetry –
synthesis
A10-3.2 [64] ●●●●●● Chemical PSMA Treatment Cell lines and mouse – Non-coding
synthesis xenograft model‡ RNAiv
PSap4#5 [36] ● Chemical PSA Diagnosis Antigen in blood Fluorescence –
PSap4#5 [37] ● Chemical PSA Diagnosis Antigen in blood Chemiluminometry –
synthesis
A9g [68] ●●● Chemical PSMA Treatment Cell lines, mouse xenograft – Directiv
synthesis model and tissue sections‡,
RNAstructure‖
A9 [69] ● Chemical PSMA Diagnosis Cell lines Photoacoustic imaging –
synthesis
S2 and R3 [26] ● Conventional PSA Diagnosis and Antigen in buffer, Mfold ‖
RT-qPCR Direct
SELEX treatment
CSC1 and ●●●● Conventional DU-145 cells and Treatment Cell lines – NIR
CSC13 [27] SELEX DU-145 stem cells irradiation
A10 [65] ●●●●●●● Chemical PSMA Treatment Cell lines‡ – Drugiv
synthesis
A10-3 [66] ●● Chemical PSMA Treatment Cell lines, mouse xenograft – Non-coding
synthesis model and tissue sections‡, RNAip
Mfold‖
(continued on next page)
3
Table 1 (continued )
Name of Refinements† Origin of Biomarker Clinical Biological samples and Diagnostic techniques Treatment
original aptamer applications assays approach
aptamer*
PAB-CoR 524 ●●● Two-hybrid AR Treatment Cell lines – Corepressor

[30] screening
A10 [54] ●●●● Chemical PSMA Treatment Antigen in buffer – Drug
synthesis
PSap4#5 [38] ●●●● Chemical PSA Diagnosis Antigen in buffer Colorimetry and –
synthesis photothermal sensing
A10 [55] ●●●● Chemical AR Diagnosis Cell lines Voltammetry –
synthesis
H1 [79] ●●●● Chemical PSA Diagnosis Antigen in buffer, blood, Fluorescence –
synthesis urine and saliva spectrometry
A10-3.2 [56] ●●● Chemical PSMA Diagnosis Cell lines and tissue SERS imaging –
synthesis (prediction and sections
monitoring)
PSap4#5 and ●●●● Chemical PSA and free PSA Diagnosis Cell lines, Mfold‖ Voltammetry –
nameless [39] synthesis
PSap4#5 [40] ●● Chemical PSA Diagnosis Antigen in buffer Impedance –
synthesis
AS1411 [81] ●● Chemical Nucleolin Treatment Cell lines, mouse xenograft – Drugiv
synthesis model and tissue sections‡
PSap4#5 [41] ●● Chemical PSA Diagnosis Blood Fluorescence –
PSap4#5 [42] ●●●● Chemical PSA Diagnosis Antigen in blood Fluorescence –
S3.1/S2.2 [82] ●● Chemical MUC1 Diagnosis Antigen in buffer and cell Voltammetry –
synthesis lines
PSap4#5 [43] ●● Chemical PSA Diagnosis Antigen in buffer Impedance –
synthesis
PSap4#5 [44] ●●● Chemical PSA Diagnosis Antigen in buffer Impedance –
synthesis
A10-3.2 [57] ●●●● Chemical PSMA Treatment Cell lines, mouse xenograft – Non-coding
synthesis model and non-human RNAiv
primates‡
DNA1 [77] ●●● Chemical PSA Diagnosis Blood Fluorescence –
A9 [70] ●●●● Chemical PSMA on CTCs Diagnosis and Cell lines and cells in blood Fluorescence NIR
synthesis treatment (rabbit) spectrometry irradiation
PSap4#5 [32] ●● Chemical PSA Diagnosis Blood Fluorescence –
XEO2 mini and ●●●●●● Cell-SELEX PC-3 and LNCaP Treatment Cell lines – Drug
XEO6 mini cells
[24]
PSap4#5 [46] ●●● Chemical PSA Diagnosis and Antigen in blood Impedance –
synthesis prognosis
SOMAmers [28] ● Proprietary SELEX Exosomal protein Diagnosis Cell lines Fluorescence –
array spectrometry
A9 CGA [71] ●●●● Chemical PSMA Diagnosis and Cell lines and mouse MRIiv Drugiv
synthesis treatment xenograft model‡
41-base A10 ●●●●●●● Chemical PSMA Treatment Cell lines – NIR
[58] synthesis irradiation
*No underlining: DNA aptamer; Simple underlining: RNA aptamer; Double underlining: peptide aptamer. ‡In vivo assays reported in addition to in vitro assays that were
used in all the articles. ‖In silico characterization. ipIntraperitoneal administration. ivIntravenous administration. †●: Dye; ●: Polymer; ●: Spacer; ●: Modified
nucleobases; ●: Gold; ●: Organic nanomaterials; ●: Hybridized oligonucleotide; ●: Solid oxide; ●: Carbon-based nanomaterial; ●: Drug. AR, androgen receptor; DBD,
DNA-binding domain; EpCAM, epithelial cell adhesion molecule; MRI, magnetic resonance imaging; MUC1, Mucin ; NIR, near-infrared; PSA, prostate specific antigen;
PSMA, prostate specific membrane antigen; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SELEX, systematic evolution of ligands by
exponential enrichment; SERS, surface-enhanced Raman scattering; VEGF, vascular endothelial growth factor.
reading and analyzing the other ones, 199 articles were found not to fit showed an appropriate experimental design. Forty-two percent of the
our eligibility criteria and were excluded (Fig. 1). The remaining 59 articles did not use relevant materials such as biological samples to test
articles were analyzed to extract data for their evaluation (Table 1). their hypotheses or theranostic platforms. Most of the articles explained
the index test in detail. When we analyzed the definition of a positive
result for those articles testing diagnostic approaches, more than a half
3.2. Quality of the selected articles
failed to report a cut off value and a few of others showed a value, but
did not specify it properly. More than one in five studies did not consider
Taking into account the total number of answered questions about
ethical issues when biological samples were used. We considered that
the quality of the selected studies, almost three quarters of them were
around 23% of the papers reported insufficiently rigorous analyses of
answered positively, 20% of questions received an unclear answer and
their data and that in 12% of them, the results section was poorly
only 2% a negative answer to the established criteria. Four percent of the
described, resulting in an uncertain analysis of data. The majority of the
answers were not applicable due to question number five (5), which was
articles displayed clearly its findings. However, more than 10% of the
specific to articles reporting aptamers for diagnostic approaches.
articles were funded by companies being susceptible to a potential
Regarding the answers for each quality question, around 45% of the
conflict of interests (Fig. 2).
articles did not state clearly the aims of the research, but all the articles
4
Fig. 1. Study flow diagram during the selection process. n, number of records.
Fig. 2. Quality assessment of the retrieved studies.
3.3. Origin of aptamers One article applied the yeast two-hybrid method to select a peptide
aptamer that bound to and suppressed the androgen receptor (AR)
Seventy-eight percent of the analyzed studies reported chemical protein [30]. Another one used complementarity of sequences to design
synthesis of aptamers, since they were previously selected and reported an aptamer against the genomic androgen response element sequence
in other papers (Fig. 3). The Cell-SELEX method, which was reported by [31]. Computational techniques such as genetic algorithms may be
eight percent of the papers [20–24], involves a cell target instead of a included after the selection process to optimize aptamers in a kind of
protein target and may be used to identify aptamers that internalize into maturation process, as in the case of the aptamer PSap4#5 [32]. Our
specific cells for targeted therapy. Conventional SELEX (protein-based results corroborate the fact that the most common aptamer selection
SELEX) appeared in seven percent of the research studies [25–28]. Two methods are protein- and cell-SELEX techniques [11]. The fact that there
articles used a proprietary SELEX technique called SOMAscan [28,29]. is no need to know the target molecule and its more native conformation
5
Fig. 3. Origin of aptamers. SELEX, systematic evolution of ligands by exponential enrichment.
– the one found in nature – on the cell-SELEX process may have made specific membrane antigen (PSMA) that have been also used in some of
this technique more common in theranostic aptamer-based applications their truncated forms such as A10-3, A10-3.2 and A9g, respectively [72].
in general and particularly in PCa. In addition, most of the aptamers PSMA is a specific biomarker for PCa that is overexpressed in PCa tis
from the retrieved studies were not selected in the reported articles. That sues. Its ability to internalize bound ligands has aroused an exceptional
indicates that aptamers are being used as targeting molecules in more interest for PCa imaging and therapy [73]. SOMAmers are slow off-rate
sophisticated tools that are under development. modified aptamers that form the affinity reagents of the proprietary
assay SOMAscan for biomarker discovery [74]. Therefore, these
aptamers may be the most promising ones for PCa due to their high
3.4. Types of aptamers characterization and wide application in several systems that are
working on their improvement. Considering the natural occurring con
In the selected articles, the use of oligonucleotide aptamers is pre dition of RNA aptamers, the relevance of PSMA as a PCa target, and the
dominantly 61% and 37% of DNA and RNA, respectively. Both types of improved affinity of A10 and A9 aptamers after their truncations, A10
aptamers have similar characteristics, but differ in terms of target and A9 aptamers stand out due to their performance in PCa theranostics.
accessibility and chemical stability. RNA aptamers are chemically un
stable due to the presence of a reactive hydroxyl group, which is
deprotonated in alkaline solutions triggering their hydrolysis. DNA 3.5. Types of ligands and biomarkers
aptamers are less reactive and relatively more stable because of the
absence of this group in deoxyribose sugar of DNA nucleotides, making During the analysis of the selected articles for this review, we
their storage, handling and processing easier and more affordable. In detected that most of them reported a target protein, while only 7%
addition, studies proved that DNA aptamers are more stable than RNA [20,21,24,27] reported target cells, namely LNCaP, 22Rv1, PC3 and DU-
aptamers in serum samples, facilitating their use in clinical settings [33]. 145, which are well-established human prostate cancer cell lines. The
In terms of production and selection, DNA aptamers are faster and first one has a high androgen sensitivity, the second one has a low
cheaper because the SELEX of RNA aptamers requires extra caution due androgen sensitivity and the others are androgen non-reliant cell lines
to chemical instability and to the fact that amplification of RNA libraries [20]. When it comes to studies that had a target protein, PSA was the
requires extra steps after every round. For these reasons, DNA aptamers most reported biomarker (46%) [8,25,26,32,37–59], maybe due to its
may be preferred in aptamer-based applications, including those for extensive use in the current clinical practice of PCa (Table 1). Some of
PCa. these studies combined PSA with other biomarkers such as epithelial cell
One of the RNA aptamers was modified with a ssDNA fragment and adhesion molecule (EpCAM) expressed on the surface of exosomes [78]
could hence be considered as a hybrid DNA-RNA aptamer. This type of and vascular endothelial growth factor (VEGF), which promotes the
hybrid aptamer is used for the treatment against PCa because this formation of blood vessels and has been studied to improve the precision
modification allows RNA aptamers to bind to their targets as well as load of PCa diagnosis [45,50]. Other biomarkers combined with PSA were
nanoparticles (NPs) for drug delivery at the same time [34]. A peptide P53 and BRCA1 genes identified by probes. These genes are important in
aptamer was also used in the analyzed studies (2%). Researchers tumor suppressing and when mutated can be indicators of prostate
selected a short amino acid stretch that binds specifically to the human tumor evolution [42].
AR in yeast and mammalian cells and is fused to a silencing domain. This PSMA was observed in 34% of the articles as a target protein
aptamer (PAB-CoR) exhibited corepressor activity by inhibiting both the [22,34,54,56–71,80]. PSMA is an integral membrane protein that
AR-mediated transactivation and the expression of its target gene PSA became an attractive target for PCa after succeeding in the detection of
[30]. metastatic PCa tumors in patients via immunoscintillography. Further
Among the most reported aptamers, PSap4#5 was found in 21 arti more, PSMA exhibits a prominent extracellular domain and naturally
cles [25,32,35–53], followed by A10, in 16 articles [22,34,54–66,79], internalizes bound ligands into the cells where it is expressed [72].
A9, in five articles [67–71] and SOMAmers, in two articles [28,29]. Three articles (5%) used the nuclear steroid receptor AR as a PCa
PSap4#5 is a well-characterized DNA aptamer ligand of PSA. A10 and biomarker, considering the important function of this receptor in the
A9 are RNA aptamer ligands of the extracellular domain of the prostate development of PCa [30,31,55]. Nucleolin is another nuclear biomarker,
6
involved in cell proliferation and overexpressed in tumor cells, that was in Raman spectroscopy [56] (Fig. 6A).
reported by one article [81]. Besides PSMA, other studies reported
membrane proteins such as MUC1, an important biomarker for cancer 3.6.2. Polymers
detection related to metastasis mechanisms and overexpressed in blood When we analyzed the aptamer refinements, polymers appeared in a
[82], and integrin α6β4, a receptor involved in tumor progression, in high percentage (13%), which are macromolecules of repeated mono
vasion and metastasis [23]. As we mentioned before, two articles used mers linked together in a three-dimensional network. From our selected
the multiplex approach SOMAscan to detect a protein array as PCa articles, we can state that polymers were able to decrease problems like
biomarkers. This technique enabled the detection of a large number of toxicity, solubility, non-specific binding and colloidal, photothermal
proteins [28,29]. These two studies together with other two [67,78] and serum instability of aptamer-targeted nanomaterials.
used proteins expressed on exosomes as biomarkers (Fig. 4). Polyethylene glycol (PEG) was the most reported polymer (41%)
found in our study [24,27,54,58,59,63–65,80]. PEG is attached to the
surface of nanocarriers to avoid nonspecific serum protein adsorption,
3.6. Aptamer refinements: Attachments and modifications
which impairs the recognition of the specific target and promotes their
clearance by the mononuclear phagocyte system. Therefore, this poly
Aptamers displayed a high variety of refinements including modifi
mer increases both the circulation time of nanocarriers in blood and the
cations to their sequence and attachments to increase their functions and
chances of target identification [84].
enhance their performance in theranostic applications. For example,
Biodegradable polymers such as polylactic acid (PLA), polylactic-co-
tumor-associated endothelial cells display an enhanced permeability
glycolic acid (PLGA) [24,54] and polycaprolactone (PCL) [63,65] were
and retention effect for nanosized materials due to their poor alignment
combined with PEG to form NPs with higher half-lives to encapsulate
around tumor-associated blood vessels. Therefore, aptamers conjugated
and control the release of therapeutic compounds, making 18% of the
to nanomaterials are able to escape from blood circulation and accu
total of polymers. Since they are amphiphilic polymers, they spontane
mulate into tumor tissues where they may also exert their theranostic
ously assemble into core–shell structures in water, which facilitates their
action because of the ineffective lymphatic drainage of these tissues
production. Furthermore, their hydrophobic core allows a high load of
[83]. We divided this complex set of improvements into ten categories to
insoluble drugs and their hydrophilic shell provides steric protection
facilitate their understanding: reporters, polymers, modified nucleo
[85]. Natural polymers such as the chitosan polysaccharide [21,25,49]
bases, spacers, gold, macromolecules, hybridized oligonucleotides, solid
and the silk fibroin protein [48] also represented 18% of the polymeric
oxides, carbon allotropes and drugs. We further explain these aptamer
modifications and were used due to their biocompatible, biomechanical,
refinements below, in order from the most to the least abundant ac
biodegradable and loading properties.
cording to the selected articles (Fig. 5).
Two studies (9%) used the polyamidoamine (PAMAM) dendrimer as
a macromolecular vector for high loading of therapeutic agents in de
3.6.1. Reporters
livery systems [64] or high loading of reporters in diagnostic systems for
Reporters for aptamer visualization and biomarker detection were
signal amplification [25]. One study used polystyrene beads called
the most common refinements (14%). They were mainly used in cell-
Dynabeads as polymeric nanocarriers [45], another study applied
based assays for preclinical diagnostic tests. Among them, fluorescent
quinone-based polymers as electrode coating [35], and another one
dyes were by far the most frequent, accounting for more than 70%. 6-
applied a polyelectrolyte multilayer of polystyrene sulfonate (PSS) and
Carboxyfluorescein (6-FAM) was the most used fluorescent dye. Redox
polyallylamine hydrochloride (PAH) on silicon nanowires [44]
probes represented 20% of the reporters [39,47,50,55,82] and were
(Fig. 6B).
used for diagnostic strategies in aptasensors with methylene blue as the
most used probe. Other types of dyes appeared only in one of the
3.6.3. Modified nucleobases
selected articles. For example, guanines were used as a chemilumines
Various chemical modifications have been introduced to prepare
cent dye [45] and 1, 4-aminothiophenol (4-ATP) was used as a reporter
Fig. 4. Biomarkers. AR, androgen receptor; MUC1, Mucin; PSA, prostate specific antigen; PSMA, prostate specific membrane antigen.
7
Fig. 5. Categories of aptamer refinements.
nucleic acids for biomedical applications, making 12% of the aptamer enable the development of sensing probes for their application in bio
refinements in our study. When thinking about aptamer modifications, sensors, diagnostic imaging and therapeutic systems. In fact, 12% of the
special attention is required to aptamers of RNA. RNA nucleotides are aptamer refinements were made with an Au nanostructure (Fig. 6E).
more prone to nuclease degradation and spontaneous hydrolysis due to Au nanomaterials act as fluorescence quenchers through fluores
their highly reactive 2′ -hydroxyl group. Several modified nucleotides cence resonance energy transfer (FRET) based on the target-induced
have been developed to stabilize RNA aptamers and still mimic their conformational change of aptamers [90]. Furthermore, Au NPs display
native counterparts [86]. tunable optical properties depending on their sizes and shapes. Their
Almost two thirds of the modified nucleotides had 2′ -fluoro groups shapes have the most influence on their optical properties affecting the
[20,22,26,34,57–59,62–66,68]. Fluorination is a particularly attractive brightness of the nanocomposites. In this way, conjugation of aptamers
modification because it can dramatically broaden the structural di to Au NPs enables the quantification of biomarkers providing a sensitive
versity of aptamers and enhance their affinity to targets [87]. Other 2′ - technique with an additional high spatial resolution for imaging [56]. In
hydroxyl substituents reported in the selected articles were 2′ -O-methyl colorimetric assays, Au NPs have a size-dependent color change prop
groups [24,68] and modified deoxyuridines (dU) such as 5-benzylami erty from red (disperse particles) to blue (aggregates) that has been used
nocarbonyl-dU (BndU), 5-naphthylmethylaminocarbonyl-dU (NapdU), to develop aptasensors [91]. Au NRs act in a very similar way to AuNPs
5-tryptaminocarbonyl-dU (TrpdU) and 5-isobutylaminocarbonyl-dU and are usually applied to enhance electron transfer in aptasensors. In
(IbdU) [28,29]. Both of these substituents represented 10% of the the case of Au electrodes, their efficient electron transfer with aptamers
modified nucleobases. The modified dUs are part of the SOMAmers, triggers signal gains or losses after their target binding that allow
which have improved binding affinity and selectivity [88]. biomarker detection [39].
Inverted deoxythymidines are another type of chemical modifica
tions of nucleotides that are incorporated at the ends of aptamers 3.6.6. Macromolecules
forming 5′ -5′ or 3′ -3′ -linked nucleotide caps. They are used mainly in Macromolecules derived from natural sources are an excellent choice
therapeutic systems because they lead to 5′ -5′ or 3′ -3′ linkages, which in terms of biocompatibility. Indeed, they were among the most used
avoid their degradation by exonucleases in blood [88]. The selected refinements for aptamers used in the PCa field (11%). Here, we
articles only described 3′ caps of inverted deoxythymidines, being 15% considered as macromolecules those that were not included in organic
of the modifications [58,62,65] (Fig. 6D). polymers or oligonucleotides.
The coenzyme biotin was one of the two most applied types of
3.6.4. Spacers macromolecules in our included articles, representing 32% of them
Nanocomposites need physical spacers of appropriate length to [25,27,34,38,44,45]. Biotin interacts non-covalently with streptavidin
reduce the steric hindrance from surrounding molecules or nano tetramers. This high-affinity interaction has allowed the use of this
materials. Thus, spacers allow adequate recognition of the target by complex in biotechnological applications to enhance targeted delivery
aptamers attached to nanocomposites [89]. Among aptamer re of loads. Streptavidin-biotin complexes conjugated to aptamers preserve
finements, spacers represented 12% of them. Eighty-five percent of the the integrity of aptamers in degrading environments [92]. For instance,
spacers were alkanes of 6, 12 or 18 carbons when attached to the 5′ or 3′ end of aptamers in applications for human
[20,27,32,35,39,40,43,46,50,51,55,56,58,63–65,68] and 15% were ol health solutions, they may avoid the activity of exonucleases decreasing
igonucleotides that were usually made of consecutively repeated nu the clearance rate of aptamers in blood circulation.
cleotides [59,71,79] (Fig. 6C). Lipids were the other most applied type of macromolecules (32%) in
our study [21,24,58,59,63,65]. They were usually combined with
3.6.5. Gold aptamers to design targeted nanostructures for diagnostic imaging and
Gold (Au) is a versatile component of nanocomposites that appeared therapeutic delivery such as phospholipid micelles or liposomes to
in the selected articles in several ways, including NPs (55%) encapsulate lipophilic drugs or imaging agents for their parenteral
[25,32,42,43,47,48,51,52,55,56,69], nanorods (NRs) (20%) delivering and cellular uptake, improving the stability of the carrier
[27,49,58,77] and as a component of electrodes (25%) system and slowing down its clearance rate.
[39,40,46,50,82]. Au nanomaterials are highly biocompatible and offer Proteins were more than a quarter of the modifications of macro
good conductive, colorimetric and nonlinear optical properties that molecules. However, they formed a very diverse group. Each protein
8
Fig. 6. Aptamer refinements. A. Reporters. B. Polymers. C. Spacers. D. Modified nucleobases. E. Gold. F. Macromolecules. G. Hybridized oligonucleotides. H. Solid
oxides. I. Carbon allotropes. J. Drugs. PAMAM, polyamidoamine; PEG, polyethylene glycol; RNA, ribonucleic acid; SOMAmers, slow off-rate modified aptamers;
ssDNA, single-stranded deoxyribonucleic acid.
9
offered different functions. For example, atelocollagen [57] and anti quantifications of tumor components such as CTCs [70] and exosomes
body anti-EpCAM [70] were combined with different aptamers to [67,78] from body fluids. They exhibited optical properties for fluo
enhance the targeting of bones and tumor epithelial cells, respectively. rescence in vitro and in vivo imaging, peroxidase-like activity for color
In order to inhibit castration-resistant PCa cell proliferation in a treat change detection, magnetic properties for biomarker capture and in vivo
ment strategy, a peptide aptamer was attached to other three protein magnetic resonance imaging, and signal amplification mediated by their
modifications: a transduction domain to internalize into cells, a good light-heat conversion for simultaneous multicolor and photo
silencing domain to inhibit both the AR-mediated transactivation and thermal sensing [34,38,71].
the expression of its target gene PSA, and a transitory glutathione-S- Nanosized TiO2 is a chemical and photochemically stable nano
transferase tag to allow its chromatographic purification [30]. material with biocompatible properties, highly available and affordable
Metal-organic compounds represented 10% of the macromolecules. [93]. TiO2 was used for signal amplification to enhance the sensitivity of
They are large self-assembled conjugate units made of highly ordered electrochemical aptasensors due to its high electron transfer ability,
crystalline materials with high porosity and large surface area. They great surface area and compatibility to attach to biomolecules such as
bind to aptamers through non-covalent interactions and contain metal aptamers and enzymes [48,76].
ions that offered redox, optical, electronic and magnetic characteristics Nanoscaled SiO2 is a semiconductor nanomaterial that may be used
suitable for biosensors. Their large volume allowed them to act as mass as an electrode substrate or covering in aptasensors. SiO2 provided an
amplifiers that proportionally amplified detection signals, while their easy surface modification, which resulted in a larger sensing area for
photo-induced electron transfer allowed them to be explored as fluo greater aptamer loading and target detection [8,39,46,50]. Further
rescence nanoquenchers [41,42], (Fig. 6F). more, SiO2 improved the photothermal stability of other nanomaterials
avoiding their melting under high intensity and extended periods of
3.6.7. Hybridized oligonucleotides therapeutic laser irradiation thanks to their thickness and fast heat
Of the total of aptamer refinements in our selected articles, 7% were dissipation, which in addition avoided aggregation-induced spectral
additional hybridized oligonucleotides partially complementary to the shifting [58].
aptamers. Forty-six percent of these hybridized oligonucleotides were
non-coding RNAs applied in therapeutic platforms and 54% of them 3.6.9. Carbon allotropes
were ssDNAs acting as linkers of nanocomposites and aptamers or as key Carbon is a chemical element able to form molecular structures of
components of enzyme-free assays (Fig. 6G). only carbon atoms called carbon allotropes. Classical carbon allotropes
Non-coding RNAs were hairpin-structure molecules delivered into are amorphous carbon, diamond and graphite. However, the most
target cells thanks to their conjugation to internalizing aptamers, whose promising carbon allotropes in theranostic applications are the novel
binding to molecular targets enabled their endocytosis. Then, non- ones involving nanomaterials derived from graphitic sheets such as
coding RNAs regulated the expression of target genes by the RNA acti graphene, carbon nanotubes (CNTs), graphene quantum-dots (GQD) and
vation process with small activating RNAs (saRNAs) [61], or the RNA fullerenes [94].
interference process with small interfering RNAs (siRNAs) [22,62,66] or Among the aptamer refinements found in our study, 6% consisted of
microRNAs (miRNAs) [57,64] in therapeutic strategies. carbon allotropes. The retrieved articles exploited the features of gra
ssDNA linkers attached to nanocomposites were combined with phene (73%), CNTs (18%) and GQD (9%) (Fig. 6I), with 80% of the
aptamers through partial hybridization with an elongated part of the articles combining these carbon allotropes with metallic NPs to enhance
aptamers that did not impair the recognition of their target molecules. the performance of aptasensors with higher surface area, catalytic
For RNA aptamers, ssDNA linkers provided higher stability, protected properties and lower limit of detection of PCa biomarkers. Among the
them from chemical modifications [34] and served as an intercalation metallic NPs, Au NPs were the most used (63%), followed by Fe3O4 NPs
site for the loading of therapeutic agents [71]. In the case of ssDNA (25%) and TiO2 NPs (12%).
aptamers, a three-way junction pocket DNA was reported. This nano Graphene is a planar graphitic sheet with a large and easily func
structure was composed of three strands of AS1411 aptamer, which tionalizable surface consisting of an sp2 hybridized carbon network with
acted as a therapeutic agent with two activities: antiproliferative activity available π electrons, which enables a high loading of other nano
due to its nucleolin targeting after internalization into target cells and materials. Thus, it may act as a substrate material in aptasensors. Half of
cytotoxic activity due to its high drug loading in its duplex regions and the articles reported the use of graphene as graphene oxide (GO)
its acidic pH-triggered drug release [81]. [38,42,70,79] and the other half as reduced graphene oxide (rGO)
ssDNAs as key components of enzyme-free assays were used in [47,51,55,76]. GO is the oxide form of graphene and rGO is derived from
different strategies, where the competition between the hybridization of GO through chemical or thermal reduction. GO displays a surface rich in
the aptamer with a partially complementary ssDNA and the binding of oxygen-containing functional groups as compared to graphene. The
this aptamer to its target allowed biomarker quantification and even removal of these groups during reduction gives rise to rGO, which also
signal amplification. For example, in colorimetric and photothermal exhibits a red-shifted absorption spectrum and higher electrical con
aptasensors [38] and in fluorescence nanoquenchers [67,77] alone or ductivity. GO showed a high optical transmittance, strong near-infrared
combined with a hybridization chain reaction [79]. (NIR) absorbance, efficient fluorescence quenching, great electron
mobility and strong affinity to ssDNA compared to dsDNA [95].
3.6.8. Solid oxides CNTs are hollow cylinders consisting of rolled-up graphitic sheets
Low-cost bulk fabrication and resistance of a wide range of pH and with high surface-to-volume ratio for a good loading and high electrical
temperatures are properties that make solid oxides attractive nano conductivity. The articles included in our study reported multi-walled
materials. Solid oxides constituted 7% of the aptamer refinements. They (MWCNTs), which comprised several concentrically graphitic sheets
can be combined with other nanomaterials to design lab-on-a-chip de interlinked via van der Waals forces [51,80].
vices such as portable aptasensors. Most of the solid oxides found in the GQDs are one or few graphitic layers with one lateral dimension
included articles were metallic oxides such as superparamagnetic iron structure up to 100 nm. They preserve a large surface area and available
oxide (Fe3O4) NPs (46%) and titanium oxide (TiO2) NPs and nanosheets π electrons, which provided an excellent photoluminescence with
(15%). However, metalloid oxides such as silicon oxide (SiO2) nanofilms resistance to photo-bleaching, electrocatalytic properties, electron
and nanowires represented 39% of the total (Fig. 6H). transfer and biocompatibility [49].
Due to their biocompatibility, high surface-area-to-volume ratio and
easy-to-conjugate targeting ligands, dyes and drugs, Fe3O4 NPs have 3.6.10. Drugs
multifunctional theranostic capabilities. Fe3O4 NPs allowed Six percent of the refinements on aptamers were drugs used as
10
antineoplastics in articles that described therapeutic platforms. The characterize the corresponding aptamers. Only the Gene Runner soft
cytostatic agent doxorubicin was the most reported drug (40%). Its red ware (Hastings Software Inc. Hastings, NY, USA, http://www.gener
fluorescence enables its detection through fluorescent microscopy or unner.net), which was reported by one article [31], characterizes and
flow cytometry. Since this chemotherapeutic compound may be uptaken additionally selects an aptamer from partially complementary sequences
by membrane-permeable non-target cells, it was carried by a targeted to the DNA binding domains of the AR receptor.
delivery system and administered at the minimum effective dose to The standard method for aptamer selection is the in vitro technique
avoid side effects. Thus, doxorubicin was able to intercalate into the SELEX. Nevertheless, less laborious techniques with higher output have
double helix of nucleic acids and establish hydrophobic interactions appeared over the last years [99]. Rather than alternative techniques to
with nanomaterials, which allowed a pH-sensitive delivery in acidic SELEX, these in silico techniques are complementary techniques. In this
conditions such as those of cancer cells and a higher drug loading way, they are combined approaches with in vitro and in silico methods. In
[21,34,71,81]. silico techniques based on computational methods such as docking,
Another drug that was reported in our study was the chemothera molecular dynamics and even machine learning allow a faster and more
peutic agent TGX221, which achieved 20% of the reported drugs. affordable selection of aptamers. These algorithms did not appear in
TGX221 is a synthetic inhibitor of phosphatidylinositol 3-kinase (PI3K), more articles because they reported aptamers previously selected and
which promotes tumorigenesis and cancer progression of tumor cells. their in silico modeling was reported in preceding articles reporting their
TGX221 displays cell membrane permeability, poor solubility in water selection.
and narrow therapeutic index that were overcome by a targeted delivery Clinical trials using theranostic application for PCa were not found in
system that specifically reached target tissues with minimal adverse the retrieved articles, which highlights the early development of these
effects [63,65]. technologies in the clinical practice of PCa. However, we expect the
Docetaxel is another chemotherapeutic drug applied in the clinical assessment of some of the most studied PCa-specific aptamers such as
treatment of PCa that appeared twice in our selected articles (20%). Due PSap4#5, A10 and A9, shortly in clinical trials.
to its hydrophobic nature, it needed to be encapsulated to internalize
target cells [24,54]. It was simultaneously co-delivered with the hy 3.8. Clinical applications
drophilic drug cisplatin, which appeared once in our study (10%). Since
they possess different solubility and mode of action, their temporally More than a half of the selected articles were diagnostic research
controlled co-delivery achieved a synergistic cytotoxic effect [54]. studies, while treatment research ones accounted for almost 34% of
Auristatin derivatives monomethyl auristatin E (MMAE) and F them. Thus, the fewest articles reported diagnostic and prognostic
(MMAF) are highly cytotoxic microtubule inhibitors. They were re research studies and theranostic research studies with 7% and 5%,
ported just once (10%) and needed a target delivery system to get into respectively (Fig. 7). All these studies showed clinically relevant targets
the cell cytoplasm and cause apoptosis [20] (Fig. 6J). and potential clinical applications that may be implemented in the
medical practice of PCa. However, there are obstacles that prevent
3.7. Types of assays aptamers from reaching their space in the general clinical practice.
Despite the advantages of aptamers against antibodies, the resistance of
The development of diagnostic, therapeutic or preventive agents for professionals to replace antibodies as tools in standard techniques is one
a particular disease may take 12 years, mainly spent on the first phases of the main obstacles. The ignorance of their existence and the lack of a
(early and late discovery) and cost around a billion dollars, mainly spent full validation for these aptamers are other obstacles [100]. These last
on the latest phases (preclinical and clinical studies). Only one of 10,000 ones depend directly on the aptamer industry and researchers and they
discovered agents is able to reach the market. Generally, the others drop could overcome that easily with more investments in scientific
from development due to efficacy and toxicity problems during pre communication and validation assays.
clinical safety studies and clinical studies. Cell-based in vitro assays are
used to optimize these agents and identify human specific risks 3.8.1. Biological samples
regarding their permeability, pharmacokinetics, bioavailability, meta The majority of the analyzed articles used prostate cell lines as bio
bolic stability and interactions with other drugs. Thus, in vitro assays logical materials to test the clinical applications of aptamer-based sys
enable the selection of candidates for more successful preclinical animal tems, followed by blood samples, mouse xenograft models, antigens in
safety studies [96]. In silico studies usually complement or precede in buffer, tissue sections, urine and saliva (Fig. 8). Regarding the cell lines
vitro assays, as they need experimental validation. Bioinformatics algo used, we only reported the prostate cell lines (Table 1), but other cell
rithms run these assays to predict the interaction of aptamers with their lines were used in some articles as controls. Among tumor prostate cell
targets offering their secondary structures, stability parameters and lines, PC-3, LNCaP, DU-145, C4–2, 22Rv1, LAPC-4 and VCaP appeared
complementarity in the case of nucleic acid targets. While in silico and in in the selected articles based on their biomarker expression levels,
vitro assays are developed in the early and late discovery phase, in vivo mainly PSMA and AR. Nonetheless, some of these cell lines were
assays are developed in the preclinical phase. Consequently, the use of in modified to change the expression of these biomarkers. Non-tumor
vivo assays would indicate a further development of these agents. Even prostate cell lines RWPE-1, PrEC, PNT2 and BPH-1 were also applied
though in vivo assays better mimic physiological conditions, we should as control cell lines.
bear in mind that studies performed in animals such as rodent or non- Within blood-derived samples, either serum spiked with the target
rodent species may fail to represent human responses because they antigen (47%) or pure serum from human blood samples (37%) was
may differ significantly in the enzymes involved in drug metabolism, gut applied in diagnostic platforms. In addition, two articles (11%) reported
physiology and pharmacokinetics [96]. human plasma [29,78] and only one (5%) reported whole rabbit blood
Therefore, our included articles showed an earlier phase of devel spiked with LNCaP cells, which imitated CTCs found in the blood of PCa
opment, as 76% reported only in vitro assays such as cell viability, serum patients [70]. Whole blood is highly degradable and requires a fast
stability, targeting assays and so forth. In vivo assays such as inhibition of processing to avoid hemolysis. Plasma corresponds to the liquid portion
tumor growth, survival and tissue distribution assays were reported by of blood after centrifugation that has been treated with an anticoagulant
24% of the selected articles with cell line-derived xenograft tumor to avoid clotting, whereas serum has the same composition but lacks
mouse models [20,22,57,59,61–66,68,71,79,80] together with in vitro fibrinogen and coagulation factors because it has not been treated [101].
assays. In silico assays of our selected studies used the software Mfold The fact that whole blood is hardly used may be explained because of its
[97], which was reported by six articles [20,26,34,39,50,66], and instability. In the case of plasma, its low use may be due to the presence
RNAstructure [98], which was reported by two articles [61,68], to of anticoagulants that interfere in the recognition of targets by their
11
Fig. 7. Clinical applications.
Fig. 8. Biological samples.
corresponding aptamers. obtaining saliva that makes this procedure a very patient-friendly one.
The selected articles reported murine xenograft models for in vivo In contrast to liquid biopsies that are easily accessible samples acquired
studies. These models were designed from immunocompromised mice by non-invasive procedures, tissue sections or tissue biopsies are ob
transplanted with cells from human PCa cell lines in the following order tained after a small surgery, an invasive procedure that was also re
from most to least used: PC-3 [59,62,71,80,81], LNCaP [57,62,63,71], ported and that is currently used in the clinical practice of PCa.
C4-2 [22,59,63], 22Rv1 [20,63] and LAPC-4 [63]. Thanks to xenograft
models, researchers were able to outline the pharmacodynamics and 3.8.2. Route of administration
pharmacokinetics of aptamer-based nanoformulas regarding tissue dis According to the articles analyzed, the most used route of adminis
tribution, maximum tolerable dose and antitumor activity. tration was intravenous, representing 75% of the studies
Antigens in buffers were used as mocked body fluids to mimic liquid [20,57,59,62,63,65,66,68,71,79,80]. The intravenous route consists of
biopsies that may load PCa-specific antigens, mainly PSA. Among body the administration of substances through injection in the vein. This
fluids, blood, seminal fluid, urine and stool are body fluids that may method allows a fast systemic circulation of the products applied and a
carry a high load of PCa components in patients affected by this disease lower concentration of them. In addition, it provides a total bioavail
due to their proximity to the prostate gland [102]. In fact, urine was the ability of pharmaceuticals and a direct distribution to all tissues of the
second body fluid most reported by the selected studies [29,67,79]. circulatory system [103,104]. Most studies used the mice’s tail vein for
However, saliva also appeared in the selected articles as a source of injection of the aptamer-targeted system [20,57,62–66,68,71,79,80].
liquid biopsy for PCa [79]. The reason for this may be the ease of Only one study [59] used the retro-orbital venous sinus of mice that is
12
located behind the eyes, in the lateral and medial corners [105]. This selected articles were based on voltammetry [35,39,47,50,52,82]
sinus is a site of safe, easy and fast application of substances because of (Fig. 9). This technique measures electric current values after varying its
its easy access to numerous blood vessels. It shows high absorption rate potential in different ways, which results in different types of voltam
and tolerance to high concentrations of solutions, as well as non- metry. The most used types were in the following order: cyclic vol
physiological pH [106]. tammetry (CV), square wave voltammetry (SWV), differential pulse
The intraperitoneal injection was used in 25% of the articles voltammetry (DPV) and differential pulse stripping voltammetry
[22,61,66], and consists of a technique of easy learning and execution, (DPSV). Interestingly, a DNA amplification technique called rolling
besides being safe and triggering little stress to the animals. This tech circle amplification (RCA) was combined with DPSV. A ssDNA primer
nique consists of the injection of substances into the peritoneal cavity, was elongated to form a long ssDNA with the assistance of a circular
which allows contact between the administered product and the sys DNA template and polymerases. This ssDNA contained thousands of
temic circulation. Although this route exhibits fast absorption of drugs, tandem repeats that were complementary to the circular template and
the pharmacokinetics of the administered drugs is similar to the oral served as a template for the formation of metal NPs, which acted as
route, occurring a high rate of metabolism of first passage. With the loss signal reporters and amplifiers. The release of metal ions in acid solu
of a portion of the injected product before reaching the bloodstream, tions were detected by DPSV to quantify a single PSA molecule after its
intraperitoneal injections would require larger quantities of the injected recognition by a specific aptamer attached to a nanocomposite together
product [107]. Therefore, this issue may be the main reason for the with the primer [52].
limited used of the intraperitoneal injection and the preference for the Thirty-seven percent of the electro-analytical methods were
intravenous one. impedimetric ones and used electrochemical impedance spectroscopy
(EIS) [40,43,44,46,48,76]. Compared with voltammetric methods, EIS
3.8.3. Diagnostic techniques uses a low excitation voltage that allows long-term monitoring and
Most of the selected papers used electro-analytical techniques in avoids electrode heating and damage preserving the biological sample
aptasensors, which are biosensors including aptamers for target detec [109]. Although voltammetry and impedance were used in several ar
tion (Fig. 9). Nowadays, biomarkers may be detected from invasive ticles to characterize and analyze the fabrication of aptasensors, only
tissue biopsies analyzed by immunohistochemistry (IHC) in the case of 19% of the articles combined them simultaneously for the detection of
proteins or fluorescence in situ hybridization (FISH) in the case of RNA or PCa biomarkers [25,49,51] (Fig. 9).
DNA. An alternative for these techniques is the non-invasive liquid bi Besides electro-analytical techniques, researchers explored other
opsies analyzed by enzyme-linked immunosorbent assays (ELISA). options. Fluorescence spectrometry appeared in 28% of the studies
However, biomarkers are detected with limited sensitivity in ELISA [28,29,32,36,41,42,67,70,74,76,77], including two articles applying
compromising the analysis of those with low concentrations. Biosensors this technique to microarray chips [28,29] and another one adding a
are reliable, fast, affordable and portable devices composed of a highly polarized filter to measure anisotropy [41]. Imaging techniques were
sensitive receptor able to recognize a specific analyte and a transducer represented in 15% of the papers. Photoacoustic imaging was the most
able to convert the detection of the analyte into a signal providing common in the selected articles [60,69], followed by magnetic reso
quantitative or semi-quantitative information [108]. The sensitivity of nance [71], ultrasound [80] that also appeared combined with fluo
these biosensors is usually enhanced through the use of emerging rescence [59] and surface-enhanced Raman scattering [56] (Fig. 9).
nanomaterials such as metal nanoparticles, polymers, carbon allotropes Imaging techniques offer in vivo information about not only the
and so on, which increase the surface area improving the conductivity of detection and level of expression of molecular biomarkers, but also
electrodes to amplify electrochemical signals [76]. about the anatomy and physiology of the tumors [110]. However, these
Forty-four percent of the electro-analytical methods reported by the techniques may be considered as a little more invasive techniques. They
Fig. 9. Diagnostic techniques. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SERS, surface-enhanced Raman scattering.
13
require the uptake of the aptamer by the patients, needing pharmaco Treatments with the direct application of an aptamer, without
kinetic and pharmacodynamic analyses. Comparing with imaging conjugation to cytotoxic agents, appeared in 17% of the treatment
techniques, electro-analytical techniques and fluorescence spectrometry research studies analyzed (Fig. 10). In these studies, aptamers bound
perform their analyses ex vivo, which makes them more patient-friendly specific targets to control cancer cell progression by inhibition of the
techniques and facilitates their implementation in PCa clinical practice. target molecule. One of the aptamers inhibited, decreased and interfered
In addition, they do not need large equipment and infrastructure. These in the expression of cancer progression genes by competing against the
may be the reasons for the greater interest in electro-analytical tech hormone response element (HRE) in its binding to the AR [31]. A9g
niques for PCa diagnosis. aptamer inhibited PSMA activity reducing metastasis [68] and the 2′ F-
Less common techniques included chemiluminometry [37,45,53] modified aptamers R3 and S2 moderated PSA activity [26]. Another one
and colorimetry [38,78], in which absorbance or transmittance may be called IDA inhibited the binding of α6β4 integrin to laminin, which ac
measured in a spectrophotometer. Colorimetry was also combined with tivates signaling pathways associated with PCa metastasis [23].
photothermal sensing [38]. Real-time apta-PCR was reported once for Three studies (13%) used direct NIR laser treatment, which
the detection of PSA, where aptamers without labeling acted as bio decreased cell viability (Fig. 10). Since radiation therapy has been
molecular recognition elements, but also as reporter molecules for PCR limited in PCa patients due to the low tolerance of the surrounding tis
amplification [26] (Fig. 9). sues, the conjugation of aptamers to radiosensitizing agents reduced
their side effects in PCa patients. Two articles used aptamers attached to
3.8.4. Treatment approaches AuNRs targeting PSMA + cells [58] and cancer stem cells [27], as these
The majority of treatments tested aptamers conjugated with NPs can transform photon energy into thermal energy inducing hyper
chemotherapeutic drugs (39%) such as doxorubicin [21,34,71,81], thermia in cells. The other one utilized an aptamer attached to GO
docetaxel [24,54], cisplatin [54], auristatin derivatives [20] and bound to the photosensitizer ICG to induce the formation of reactive
TXG122 [63,65] (Fig. 10). Chemotherapy is a potent treatment, but as oxygen species (ROS) inside PSMA + cells after exposure to laser irra
chemotherapeutic agents have side effects and interfere in patients’ diation [70].
quality of life [34], target drug delivery systems are needed. Indeed, our Most treatment approaches for PCa conjugated the aptamers to
selected therapeutic studies evaluated aptamers conjugated with drugs therapeutic agents, evidencing that just a small portion of them interact
commonly used in PCa treatment that are toxic to other cells. directly with therapeutic targets and inhibit them. Although chemo
Thirty-one percent of our retrieved articles assessed aptamers con therapy was the most common approach, the development of tran
jugated to transcriptional modifiers being most of them non-coding scriptional modifiers seems to be a growing area in aptamer-based
RNAs such as saRNAs, siRNAs and miRNAs and including the small treatments for PCa.
ubiquitin-related modifier (SUMO) protein (Fig. 10). Transcriptional
modifiers associated with aptamers increase, decrease or even silence 4. Conclusions
gene expression. One article tried a saRNA to increase DPYSL3v2 gene
expression, which was considered as a metastatic suppressor in PCa Current epidemiology of PCa indicates that new PCa diagnostic and
[61]. Differently, two siRNAs were used to silence EGFR and survivin therapeutic modalities are necessary. Studies worldwide that used
oncogenes inhibiting multiple signaling pathways [22]. Other two aptamers for clinical applications in PCa over the last 10 years were
studies used a specific siRNA to knockdown DNAPK gene, which led to identified. They showed the tendencies for the use of aptamers in this
the inhibition of a related double-strand DNA break (DSB) repair area. In general, well-established aptamers for PCa were modified with a
pathway and to the consequent sensitization of target PSMA-positive high variety of refinements including modifications to their sequence
cells to ionizing radiation [62,66]. Another transcriptional silencer is and attachments to increase their functions and enhance their perfor
the corepressor SUMO, whose mutant SUMOG97A was used to suppress mance in theranostic applications. In this way, newly selected aptamers
the AR, known as one of the principal promoters of PCa proliferation for PCa were scarce. The target molecules of aptamers for PCa were
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The use of aptamers in prostate cancer: A systematic review of

1. Introduction as much [3,4].

apt [75] – Chemical PSA Diagnosis Antigen in blood Fluorescence –

PAB-CoR 524 ●●● Two-hybrid AR Treatment Cell lines – Corepressor

Fig. 2. Quality assessment of the retrieved studies.

Fig. 3. Origin of aptamers. SELEX, systematic evolution of ligands by exponential enrichment.

Fig. 5. Categories of aptamer refinements.

Fig. 7. Clinical applications.

Fig. 8. Biological samples.

Fig. 10. Treatment approaches. NIR, near infrared.

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