Papers by Maria Paula Cabral Campello
Journal of Inorganic Biochemistry, 2017
Aiming to explore alternative mechanisms of cellular uptake and cytotoxicity, we have studied a n... more Aiming to explore alternative mechanisms of cellular uptake and cytotoxicity, we have studied a new family of copper(II) complexes (CuL 1-CuL 4) with bis(thiosemicarbazone) (BTSC) ligands containing pendant protonable cyclic amines (morpholine and piperidine). Herein, we report on the synthesis and characterization of these new complexes, as well as on their biological performance (cytotoxic activity, cellular uptake, protein and DNA binding), in comparison with the parental Cu II ATSM (ATSM = diacetyl-bis(N4-methylthiosemicarbazonate) complex without pendant cyclic amines. The new compounds have been characterized by a range of analytical techniques including ESI-MS, IR spectroscopy, cyclic voltammetry, reverse-phase HPLC and X-ray spectroscopy. In vitro cytotoxicity studies revealed that the copper complexes are cytotoxic, unlike the corresponding ligands, with a similar potency to that of CuATSM. Unlike CuATSM, the new complexes were able to circumvent cisplatin cross-resistance. The presence of the protonable cyclic amines did not lead to an enhancement of the interaction of the complexes with human serum albumin or calf thymus DNA. However, CuL 1-CuL 4 showed a remarkably augmented cellular uptake compared with CuATSM, as proved by uptake, internalization and externalization studies that were performed using the radioactive congeners 64 CuL 1-64 CuL 4. The enhanced cellular uptake of CuL 1-CuL 4 indicates that this new family of Cu II BTSC complexes deserves to be further evaluated in the design of metallodrugs for cancer theranostics.
International Journal of Molecular Sciences, 2022
Despite some progress, the overall survival of patients with glioblastoma (GBM) remains extremely... more Despite some progress, the overall survival of patients with glioblastoma (GBM) remains extremely poor. In this context, there is a pressing need to develop innovative therapy strategies for GBM, namely those based on nanomedicine approaches. Towards this goal, we have focused on nanoparticles (AuNP-SP and AuNP-SPTyr8) with a small gold core (ca. 4 nm), carrying DOTA chelators and substance P (SP) peptides. These new SP-containing AuNPs were characterized by a variety of analytical techniques, including TEM and DLS measurements and UV-vis and CD spectroscopy, which proved their high in vitro stability and poor tendency to interact with plasma proteins. Their labeling with diagnostic and therapeutic radionuclides was efficiently performed by DOTA complexation with the trivalent radiometals 67Ga and 177Lu or by electrophilic radioiodination with 125I of the tyrosyl residue in AuNP-SPTyr8. Cellular studies of the resulting radiolabeled AuNPs in NKR1-positive GBM cells (U87, T98G and U3...
European Journal of Pharmaceutical Sciences
Ln(III) and Cu(II) complexes are utilized in medicine and preclinical research as magnetic resona... more Ln(III) and Cu(II) complexes are utilized in medicine and preclinical research as magnetic resonance, optical or nuclear probes for diagnostics and/or for cancer treatment. For biomedical applications, such complexes should exhibit a high thermodynamic stability as well as kinetic inertness under physiological conditions. Thus, knowledge of their thermodynamic/kinetic properties (e.g. dissociation rate constants for an estimation of kinetic inertness) is important to evaluate their use in these applications. Here, thermodynamic and kinetic properties of Cu(II), Ce(III) and Eu(III) complexes with macrocyclic cyclen-based ligands where acetate pendant arms were substituted by phosphonates (H4dota, H5do3ap, trans-H6do2a2p, H7doa3p, H8dotp) [1–4] are presented. In addition, the influence of substitution of one acetate/phosphonate of (H2do2a/H3do3a/H4dota/H5do3ap derivatives) and/or all phosphonate pendant arms (H8dotp derivatives) was also studied [5-7]. These results show that the kine...
To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors,... more To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors, we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types. The DTPA derivatives were unable to provide a stable coordination of Ga, and therefore, the functionalization with the BBN analogues was pursued for the DOTA-containing AuNPs. The DOTA-coated AuNPs were functionalized with BBN[7−14] using a unidentate cysteine group or a bidentate thioctic group to attach the peptide. AuNPs functionalized with thioctic-BBN displayed the highest in vitro cellular internalization (≈ 25%, 15 min) in gastrin releasing peptide (GRP) receptor expressing cancer cells. However, these results fail to translate to in vivo tumor uptake. Biodistribution studies following intravenous (IV) and intraperit...
European Journal of Inorganic Chemistry
Cancers
Cervical cancer is one of the most common cancers and is one of the major cause of deaths in wome... more Cervical cancer is one of the most common cancers and is one of the major cause of deaths in women, especially in underdeveloped countries. The patients are usually treated with surgery, radiotherapy, and chemotherapy. However, these treatments can cause several side effects and may lead to infertility. Another concerning gynecologic cancer is endometrial cancer, in which a high number of patients present a poor prognosis with low survival rates. AS1411, a DNA aptamer, increases anticancer therapeutic selectivity, and through its conjugation with gold nanoparticles (AS1411-AuNPs) it is possible to improve the anticancer effects. Therefore, AS1411-AuNPs are potential drug carriers for selectively delivering therapeutic drugs to cervical cancer. In this work, we used AS1411-AuNPs as a carrier for an acridine orange derivative (C8) or Imiquimod (IQ). The AS1411 aptamer was covalently bound to AuNPs, and each drug was associated via supramolecular assembly. The final nanoparticles prese...
Nanomaterials
Root extracts from Danube Delta Nymphaea alba were used to prepare gold nanoparticles (AuNPRn) by... more Root extracts from Danube Delta Nymphaea alba were used to prepare gold nanoparticles (AuNPRn) by reducing HAuCl4 at different pHs (6.4–8.4) using ultrasonic irradiation: an easy, cheap, eco-friendly and green approach. Their antibacterial and anticancer activities were evaluated against Staphylococcus aureus and Escherichia coli, and A2780 ovarian cancer cells, respectively. The AuNPRn were characterized concerning their phytoconstituents (polyphenols, flavonoids and condensed tannins) and gold content. All of the nanoparticles were negatively charged. AuNPRn exhibited a hydrodynamic size distribution ranging from 32 nm to 280 nm, with the larger nanoparticles being obtained with an Au/root extract ratio of 0.56, pH 7 and 10 min of sonication (AuNPR1), whereas the smallest were obtained with an Au/root extract ratio of 0.24, pH 7.8 and 40 min of sonication (AuNPR4). The TEM/SEM images showed that the AuNPRn had different shapes. The ATR-FTIR indicated that AuNPRn interact mainly wi...
Materials
In the Last decades, nanotechnology has provided novel and alternative methodologies and tools in... more In the Last decades, nanotechnology has provided novel and alternative methodologies and tools in the field of medical oncology, in order to tackle the issues regarding the control and treatment of cancer in modern society. In particular, the use of gold nanoparticles (AuNPs) in radiopharmaceutical development has provided various nanometric platforms for the delivery of medically relevant radioisotopes for SPECT/PET diagnosis and/or radionuclide therapy. In this review, we intend to provide insight on the methodologies used to obtain and characterize radiolabeled AuNPs while reporting relevant examples of AuNPs developed during the last decade for applications in nuclear imaging and/or radionuclide therapy, and highlighting the most significant preclinical studies and results.
Nucleic Acid Therapeutics
Herein, we report, for the first time, the screening of several ligands in terms of their ability... more Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.
Materials
Gold nanoparticles (AuNPs) are interesting for the design of new cancer theranostic tools, mainly... more Gold nanoparticles (AuNPs) are interesting for the design of new cancer theranostic tools, mainly due to their biocompatibility, easy molecular vectorization, and good biological half-life. Herein, we report a gold nanoparticle platform as a bimodal imaging probe, capable of coordinating Gd3+ for Magnetic Resonance Imaging (MRI) and 67Ga3+ for Single Photon Emission Computed Tomography (SPECT) imaging. Our AuNPs carry a bombesin analogue with affinity towards the gastrin releasing peptide receptor (GRPr), overexpressed in a variety of human cancer cells, namely PC3 prostate cancer cells. The potential of these multimodal imaging nanoconstructs was thoroughly investigated by the assessment of their magnetic properties, in vitro cellular uptake, biodistribution, and radiosensitisation assays. The relaxometric properties predict a potential T1- and T2- MRI application. The promising in vitro cellular uptake of 67Ga/Gd-based bombesin containing particles was confirmed through biodistrib...
Inorganica Chimica Acta
Abstract A new mixed lanthanide(III) complex [LaNd(μ2-DPY)(μ4-SO4)2(Et3N)]Br2·2H2OMeOH (La-Nd-DPY... more Abstract A new mixed lanthanide(III) complex [LaNd(μ2-DPY)(μ4-SO4)2(Et3N)]Br2·2H2OMeOH (La-Nd-DPY) was obtained by the reaction of N,N′-diphenacyl-4,4′-dipyridinium dibromide (DPB) with a mixture of La(III) and Nd(III) sulfates in a 2:1 M ratio, in the presence of triethylamine (Et3N). The method used for the synthesis of La-Nd-DPY promoted the in situ transformation of the pro-ligand DPB into the dipyridinium ylide-based ligand. Fourier transform infrared spectroscopy (FTIR), elemental analysis (EA), thermogravimetric analysis (TGA) and mass spectrometry (MS) concurred to propose a linear polymeric structure for La-Nd-DPY, in which both La(III) and Nd(III) ions are six-coordinated. Cyclic voltammetry was also used to assess the redox potential of the mixed complex. Scanning electron microscopy (SEM) showed quite uniform and homogeneous fibrillary net-like morphology and also confirmed, by means of EDX analysis, the presence of both lanthanide (III) ions in the mixed complex. UV–vis absorption spectroscopy demonstrated that the mixed Ln complex is stable up to 1 h in DMSO and up to 72 h under the physiological conditions used for cell culture. The cytotoxic activity of La-Nd-DPY in cancer cell lines of ovarian (A2780), breast (MCF7) and prostate (PC3) origins and in multicellular tumor spheroids derived from PC3 cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and acid phosphatase (APH) assays. Compared to cisplatin this new complex showed improved toxicity, with IC50 values at least 10 times higher, even at 24 h of exposure. The cytotoxic effects seemed to be mediated by reactive oxygen species (ROS) generation but not apoptosis, confirmed by a caspase-3/7 activation and Hoechst nuclei staining assays. The mechanism of cell death observed on the cytotoxicity assays is probably due to other pathways of cell death, which deserve to be further investigated.
Nuclear Medicine and Biology
European Journal of Pharmaceutics and Biopharmaceutics
Nucleic acid aptamers have emerged as an attractive class of carrier molecules due to their abili... more Nucleic acid aptamers have emerged as an attractive class of carrier molecules due to their ability to bind with high affinity to specific ligands; their high chemical flexibility; as well as tissue penetration capability. RNA G-quadruplex (rG4) sequences have been described as structures with high stability and selectivity towards cancer cells. Recently, precursor microRNAs (pre-miRNAs) have been described as new G4 forming molecules. Surface nucleolin (NCL) is a known target of aptamer G4 AS1411 and is overexpressed on prostate cancer cells when compared with normal cells. We have shown that the sequence 5' GGGAGGGAGGGACGGG 3' found in pre-miR-149 forms a rG4 parallel structure, which can bind NCL. Also, another rG4 sequence with a longer loop was evaluated in terms of G4 formation, stabilization and binding affinity to NCL. Both rG4s sequences were studied as supramolecular carriers for the cancer-selective delivery of acridine ligand C8. The rG4s-C8 complexes showed high affinity (KD =10-6 M) and stabilization (Tm >30 °C). The affinity of the rG4s-C8 complexes against NCL was in the low nanomolar range, indicating that C8 did not affect NCL binding. Noteworthy, the short loop rG4-C8 complex showed selective antiproliferative effects in prostate cancer cells when compared with normal prostatic cells. The stability and nuclease resistance of rG4 and rG4-C8 complex were evaluated in biological conditions and revealed the maintenance of G4 structure and complex stability. Furthermore, confocal microscopy studies confirmed the potential of rG4s-C8 complexes in the targeting of prostate cancer cells. Overall, it is here demonstrated that the rG4 found in pre-miR-149 can be used as a cancer-selective delivery carrier of C8 to prostate cancer cells.
International Journal of Pharmaceutics
Nucleic acid aptamers can specifically bind to target molecules on the cell membrane that mediate... more Nucleic acid aptamers can specifically bind to target molecules on the cell membrane that mediate their entrance into the cells. Their small size, high binding affinity, specificity, good biocompatibility, stability and low immunogenicity make them ideal drug delivery systems for cancer therapy. These biopharmaceuticals have potential for the delivery of anticancer compounds to diseased tissues, increasing their effectiveness while mitigating the off-target toxicity towards healthy cells. Herein, we have studied two quadruplex-forming DNA sequences derived from the nucleolin-targeted aptamer AS1411 as supramolecular carriers for the cancer-selective delivery of acridine orange derivatives (C3, C5 and C8) in cervical cancer cells. The devised delivery strategy relied on the non-covalent association of the acridine derivatives and the G-quadruplex (G4) structures. This association is done with a high binding strength, as suggested by the obtained KD values in the 10-6 - 10-7 M range, leading to the thermal stabilization of the G4 structures, particularly for C8. The stability of the resulting supramolecular conjugates was evaluated in fetal bovine serum, which proved their resistance against serum nucleases up to 48 h. Previous studies showed that the tested acridine orange derivatives were cytotoxic towards cervical cancer cells (HeLa) and non-malignant cells. However, when conjugated to AS1411 derivatives, the cytotoxicity of the free ligands towards non-malignant cells was restrained. Furthermore, conjugated C3 showed an enhanced cytotoxicity against HeLa cancer cells. Confocal microscopy indicated that both G4 sequences appear to colocalize with nucleolin, suggesting their ability to recognize and bind nucleolin on the cell surface. Additionally, the results confirmed the internalization of these delivery systems into HeLa cancer cells and their sustained cell trafficking, although being able to dissociate intracellularly to deliver C8 to the nucleoli. Overall, we showed that AS1411-derived G4s can be used as a potential cancer drug delivery system for cervical cancer.
Scientific Reports
AS1411 is a G-rich DNA oligonucleotide that functions as an aptamer of the protein nucleolin, fou... more AS1411 is a G-rich DNA oligonucleotide that functions as an aptamer of the protein nucleolin, found at high levels on the surface of cancer cells but not on the surface of normal cells. Herein, we have studied AS1411 as a supramolecular carrier for the delivery of an acridine-based G-quadruplex ligand, C 8 , to HeLa cancer cells. Two AS1411 derivatives, LNA-AS1411 and U-AS1411, were also tested, in an attempt to compare AS1411 pharmacological properties. The results showed that AS1411-C 8 complexation was made with great binding strength and that it lowered the ligand's cytotoxicity towards non-malignant cells. This effect was suggested to be due to a decreased internalization of the complexed versus free C 8 as shown by flow cytometry. The AS1411 derivatives, despite forming a stable complex with C 8 , lacked the necessary tumour-selective behaviour. the binding of C 8 to AS1411 G-quadruplex structure did not negatively affect the recognition of nucleolin by the aptamer. The AS1411-C 8 repressed c-MYC expression at the transcriptional level, possibly due to C 8 ability to stabilize the c-MYC promoter G-quadruplexes. Overall, this study demonstrates the usefulness of AS1411 as a supramolecular carrier of the G-quadruplex binder C 8 and the potential of using its tumour-selective properties for the delivery of ligands for cancer therapy. AS1411 is a synthetic 26-base DNA aptamer previously discovered by Bates and co-workers as an agent targeting the nucleolin protein with high affinity and specificity, eliciting a potent antiproliferative effect on a variety of cell lines 1,2. Nucleolin is a multifunctional protein playing essential roles in cell survival, growth and proliferation. The protein is localized primarily in the nucleus of normal cells, while on cancer cells it is also present in the cytoplasm and at the cell surface 3. This property confers a tumour-selective behaviour to AS1411 which targets preferentially the external domain of surface nucleolin of cancer cells. This oligonucleotide reached Phase 2b clinical trials for acute myeloid leukaemia and renal cell carcinoma; however, despite a good tolerance and safety profile, the trial was terminated due to suboptimal pharmacokinetics (rapid clearance) and low potency 2. The mechanism of the cytotoxicity of AS1411 is still under debate and multiple nucleolin-dependent and independent biological effects have been described 2. Recently, methuosis has been proposed as the likely cell death mechanism induced by AS1411, which is characterized by altered cell morphology and hyperstimulation of macropinocytosis combined with perturbed vesicle trafficking 4. AS1411 is able to fold into a highly polymorphic G-quadruplex structure which makes it more stable against the serum nucleases and pH fluctuations, with increased cellular uptake efficacy 1,2. Regardless of the clinical application of AS1411 being hindered, its tumour-selective properties have increasingly motivated its study as a drug delivery system of therapeutic agents and imaging probes. The variety of applications range from nanoparticle-linked AS1411 formulations to covalent/noncovalent drug conjugates 2. The diversity of cargoes that have been efficiently delivered by AS1411 extend to G-quadruplex ligands as its own
Dalton Transactions
Herein we report the synthesis, characterization, photophysical and biological evaluation of [Ln(... more Herein we report the synthesis, characterization, photophysical and biological evaluation of [Ln(DBM)3(RPhen)] (Ln= Sm, R= H; Ln= Sm, Eu, Tb, R= 5-NH2) complexes stabilized by three β-diketonate units (DBM) and...
Organic & Biomolecular Chemistry
DNA aptamers represent a way to target cancer cells at a molecular level and continue to be devel... more DNA aptamers represent a way to target cancer cells at a molecular level and continue to be developed with a view to improve treatment and imaging in cancer medicine. AT11-L0,...
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, Jan 24, 2018
The development of pharmacologically active compounds based on bis(thiosemicarbazones) (BTSC) and... more The development of pharmacologically active compounds based on bis(thiosemicarbazones) (BTSC) and on their coordination to metal centers constitutes a promising field of research. We have recently explored this class of ligands and their Cu(II) complexes for the design of cancer theranostics agents with enhanced uptake by tumoral cells. In the present work, we expand our focus to aliphatic and aromatic BTSC Zn(II) complexes bearing piperidine/morpholine pendant arms. The new complexes ZnL-ZnL were characterized by a variety of analytical techniques, which included single-crystal X-ray crystallography for ZnL and ZnL. Taking advantage of the fluorescent properties of the aromatic complexes, we investigated their cellular uptake kinetics and subcellular localization. Furthermore, we tried to elucidate the mechanism of action of the cytotoxic effect observed in human cancer cell line models. The results show that the aliphatic complexes (ZnL and ZnL) have a symmetrical structure, while...
Uploads
Papers by Maria Paula Cabral Campello