Assessing the gene silencing potential of AuNP-based approaches on conventional 2D cell culture versus 3D tumor spheroid.

Three-dimensional (3D) cell culture using tumor spheroids provides a crucial platform for replicating tissue microenvironments. However, effective gene modulation via nanoparticle-based transfection remains a challenge, often facing delivery hurdles. Gold nanoparticles (AuNPs) with their tailored synthesis and biocompatibility, have shown promising results in two-dimensional (2D) cultures, nevertheless, they still require a comprehensive evaluation before they can reach its full potential on 3D models. While 2D cultures offer simplicity and affordability, they lack physiological fidelity. In contrast, 3D spheroids better capture in vivo conditions, enabling the study of cell interactions and nutrient distribution. These models are essential for investigating cancer behavior, drug responses, and developmental processes. Nevertheless, transitioning from 2D to 3D models demands an understanding of altered internalization mechanisms and microenvironmental influences. This study assessed ASO-AuNP conjugates for silencing the c-MYC oncogene in 2D cultures and 3D tumor spheroids, revealing distinctions in gene silencing efficiency and highlighting the microenvironment's impact on AuNP-mediated gene modulation. Herein, we demonstrate that increasing the number of AuNPs per cell by 2.6 times, when transitioning from a 2D cell model to a 3D spheroid, allows to attain similar silencing efficiencies. Such insights advance the development of targeted gene therapies within intricate tissue-like contexts.

Epidermal growth factor alters silica nanoparticle uptake and improves gold-nanoparticle-mediated gene silencing in A549 cells.

Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs. Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC. Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC. Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.

Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples.

The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor's temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes.

Engineering gold nanoparticles for molecular diagnostics and biosensing.

Advances in nanotechnology and medical science have spurred the development of engineered nanomaterials and nanoparticles with particular focus on their applications in biomedicine. In particular, gold nanoparticles (AuNPs) have been the focus of great interest, due to their exquisite intrinsic properties, such as ease of synthesis and surface functionalization, tunable size and shape, lack of acute toxicity and favorable optical, electronic, and physicochemical features, which possess great value for application in biodetection and diagnostics purposes, including molecular sensing, photoimaging, and application under the form of portable and simple biosensors (e.g., lateral flow immunoassays that have been extensively exploited during the current COVID-19 pandemic). We shall discuss the main properties of AuNPs, their synthesis and conjugation to biorecognition moieties, and the current trends in sensing and detection in biomedicine and diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Light Triggered Enhancement of Antibiotic Efficacy in Biofilm Elimination Mediated by Gold-Silver Alloy Nanoparticles.

Bacterial biofilm is a tri-dimensional complex community of cells at different metabolic stages involved in a matrix of self-produced extracellular polymeric substances. Biofilm formation is part of a defense mechanism that allows the bacteria to survive in hostile environments, such as increasing resistance or tolerance to antimicrobial agents, causing persistent infections hard to treat and impair disease eradication. One such example is bovine mastitis associated with Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), whose worldwide health and economic impact is on the surge. As such, non-conventional nanobased approaches have been proposed as an alternative to tackle biofilm formation and to which pathogenic bacteria fail to adapt. Among these, metallic nanoparticles have gained significant attention, particularly gold and silver nanoparticles, due to their ease of synthesis and impact against microorganism growth. This study provides a proof-of-concept investigation into the use of gold-silver alloy nanoparticles (AuAgNPs) toward eradication of bacterial biofilms. Upon visible light irradiation of AuAgNPs there was considerable disturbance of the biofilms' matrix. The hindering of structural integrity of the biofilm matrix resulted in an increased permeability for entry of antibiotics, which then cause the eradication of biofilm and inhibit subsequent biofilm formation. Additionally, our results that AuAgNPs inhibited the formation of SDSD biofilms via distinct stress pathways that lead to the downregulation of two genes critical for biofilm production, namely, brpA-like encoding biofilm regulatory protein and fbpA fibronectin-binding protein A. This study provides useful information to assist the development of nanoparticle-based strategies for the active treatment of biofilm-related infections triggered by photoirradiation in the visible.

Drug delivery nanosystems targeted to hepatic ischemia and reperfusion injury.

Hepatic ischemia and reperfusion injury (IRI) is an acute inflammatory process that results from surgical interventions, such as liver resection surgery or transplantation, or hemorrhagic shock. This pathology has become a severe clinical issue, due to the increasing incidence of hepatic cancer and the high number of liver transplants. So far, an effective treatment has not been implemented in the clinic. Despite its importance, hepatic IRI has not attracted much interest as an inflammatory disease, and only a few reviews addressed it from a therapeutic perspective with drug delivery nanosystems. In the last decades, drug delivery nanosystems have proved to be a major asset in therapy because of their ability to optimize drug delivery, either by passive or active targeting. Passive targeting is achieved through the enhanced permeability and retention (EPR) effect, a main feature in inflammation that allows the accumulation of the nanocarriers in inflammation sites, enabling a higher efficacy of treatment than conventional therapies. These systems also can be actively targeted to specific compounds, such as inflammatory markers and overexpressed receptors in immune system intermediaries, allowing an even more specialized therapy that have already showed encouraging results. In this manuscript, we review drug delivery nanosystems designed for hepatic IRI treatment, addressing their current state in clinical trials, discussing the main hurdles that hinder their successful translation to the market and providing some suggestions that could potentially advance their clinical translation.

Tackling Multidrug Resistance in Streptococci - From Novel Biotherapeutic Strategies to Nanomedicines.

The pyogenic streptococci group includes pathogenic species for humans and other animals and has been associated with enduring morbidity and high mortality. The main reason for the treatment failure of streptococcal infections is the increased resistance to antibiotics. In recent years, infectious diseases caused by pyogenic streptococci resistant to multiple antibiotics have been raising with a significant impact to public health and veterinary industry. The rise of antibiotic-resistant streptococci has been associated to diverse mechanisms, such as efflux pumps and modifications of the antimicrobial target. Among streptococci, antibiotic resistance emerges from previously sensitive populations as result of horizontal gene transfer or chromosomal point mutations due to excessive use of antimicrobials. Streptococci strains are also recognized as biofilm producers. The increased resistance of biofilms to antibiotics among streptococci promote persistent infection, which comprise circa 80% of microbial infections in humans. Therefore, to overcome drug resistance, new strategies, including new antibacterial and antibiofilm agents, have been studied. Interestingly, the use of systems based on nanoparticles have been applied to tackle infection and reduce the emergence of drug resistance. Herein, we present a synopsis of mechanisms associated to drug resistance in (pyogenic) streptococci and discuss some innovative strategies as alternative to conventional antibiotics, such as bacteriocins, bacteriophage, and phage lysins, and metal nanoparticles. We shall provide focused discussion on the advantages and limitations of agents considering application, efficacy and safety in the context of impact to the host and evolution of bacterial resistance.

Water safety screening via multiplex LAMP-Au-nanoprobe integrated approach.

Contaminated water resources remain a major global concern regarding public health. The majority of water safety protocols include indicators of microbial contamination to evaluate the potential risk to public health and are key elements of quality guidelines. Among these, markers for total coliforms and fecal coliforms are strong indicators of co-contamination with other pathogens. Traditional methods, recurring to slow and cumbersome culture-based approaches, have been gradually replaced by molecular methods, capable of faster and more specific screening. These are usually PCR-based methods that may allow for multiple pathogen detection but require dedicated laboratory equipment, hindering the rapid on-site assessment. Here, we used a multiplex Loop-Mediated Isothermal Amplification (mLAMP) strategy for the amplification of two markers associated with the contamination by total and fecal coliforms (e.g. Escherichia coli) - lacZ and uidA genes, respectively - thus allowing for single tube multiplex detection. The mLAMP products were then subject to an Au-nanoprobe colorimetric detection assay for precise discrimination of targets. This approach was validated in 22 water samples that were also screened for the presence of lacZ and uidA using standard and quantitative PCR, with the capability for discriminating the contamination level, e.g. a semi-quantitative evaluation of water quality.

Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers.

Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in "Shrinky-dinks" polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.

RNA Quantification Using Noble Metal Nanoprobes: Simultaneous Identification of Several Different mRNA Targets Using Color Multiplexing and Application to Chronic Myeloid Leukemia Diagnostics.

Nanotechnology provides new tools for gene expression analysis that allow for sensitive and specific characterization of prognostic signatures related to cancer. Cancer is a complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus allows for a more accurate indication into the degree of cancerous activity than either locus alone. Metal nanoparticles have been widely used as labels for in vitro identification and quantification of target sequences.Here we describe the synthesis of nanoparticles with different noble metal compositions in an alloy format that are then functionalized with thiol-modified ssDNA (nanoprobes). We also show how such nanoprobes are used in a non-cross-linking colorimetric method for the direct detection and quantification of specific mRNA targets, without the need for enzymatic amplification or reverse-transcription steps. The different metals in the alloy provide for distinct absorption spectra due to their characteristic plasmon resonance peaks. The color multiplexing allows for simultaneous identification of different mRNA targets involved in cancer development. A comparison of the absorption spectra of the nanoprobe mixtures taken before and after induced aggregation of metal nanoparticles allows to both identify and quantify each mRNA target. We describe the use of gold and gold-silver alloy nanoprobes for the development of the non-cross-linking method to detect a specific BCR-ABL fusion gene (e.g., e1a2 and e14a2) mRNA target associated with chronic myeloid leukemia (CML) using 10 ng/µL of unamplified total human RNA. Additionally, we demonstrate the use of this approach for the direct diagnostics of CML. This simple methodology takes less than 50 min to complete after total RNA extraction with comparable specificity and sensitivity to the more commonly used methods.

Paper-Based SERS Platform for One-Step Screening of Tetracycline in Milk.

Throughout the last decade, the expansion of food testing has been gradually moving towards ordinary high throughput screening methods performed on-site. The demand for point-of-care testing, able to distinguish molecular signatures with high accuracy, sensitivity and specificity has been significantly increasing. This new requirement relies on the on-site detection and monitorization of molecular signatures suitable for the surveillance of food production and processing. The widespread use of antibiotics has contributed to disease control of livestock but has also created problems for the dairy industry and consumers. Its therapeutic and subtherapeutic use has increased the risk of contamination in milk in enough concentrations to cause economic losses to the dairy industry and have a health impact in highly sensitive individuals. This study focuses on the development of a simple Surface-Enhanced Raman Spectroscopy (SERS) method for fast high throughput screening of tetracycline (TET) in milk. For this, we integrate a paper-based low-cost, fully recyclable and highly stable SERS platform, with a minimal sample preparation protocol. A two-microliter sample of milk solutions spiked with TET (from 0.01 to 1000 ppm) is dried on a silver nanoparticle coated cardboard substrate and measured via a Raman spectrophotometer. The SERS substrate showed to be extremely stable with a shelf life of several months. A global spectrum principal component analysis approach was used to test all the detected vibrational modes and their correlation with TET concentration. Peak intensity ratios (455 cm-1/1280 cm-1 and 874 cm-1/1397 cm-1) were found to be correlated with TET concentrations in milk, achieving a sensitivity as low as 0.1 ppm. Results indicate that this SERS method combined with portable Raman spectrometer is a potential tool that can be used on-site for the monitoring of TET residues and other antibiotics.

Antibody modified gold nanoparticles for fast colorimetric screening of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation and one of the main causes of chronic disability worldwide with high prevalence in the ageing population. RA is characterized by autoantibody production, synovial inflammation and bone destruction, and the most accepted biomarker is rheumatoid factor (RF) autoantibodies. In this work, we developed a low-cost approach for the detection and quantification of the RF marker. This colorimetric immunosensor is based on gold nanoprobe crosslinking that results in extensive aggregation in the presence of the pentameric IgM RF. Aggregation of the nanoconjugates yields a color change from red to purple that can be easily observed by the naked eye. The interaction between nanoconjugates and the specific target was confirmed via dynamic light scattering (DLS), Raman spectroscopy and atomic force microscopy (AFM) imaging. This conceptual system shows a LOD of 4.15 UA mL-1 IgM RF (clinical threshold is set for 20 IU mL-1). The one-step biosensor strategy herein proposed is much faster than conventional detection techniques, without the need for secondary antibodies, additional complex washing or signal amplification protocols. To the best of our knowledge this is the first report on target induced aggregation of gold nanoprobes for quantitative colorimetric autoantibody detection.

A novel BCR-ABL1 mutation in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) represents the most common genetic subtype of adult ALL (20%-30%) and accounts for approximately 50% of all cases in the elderly. It has been considered the subgroup of ALL with the worst outcome. The introduction of tyrosine kinase inhibitors (TKIs) allows complete hematologic remission virtually in all patients, with improved disease-free survival and overall survival. Nevertheless, the emergence of resistant mutations in BCR-ABL1 may require different TKI strategies to overcome the patient's resistance and disease relapse. Here, we report a Ph+B-ALL case with persistent minimal residual disease (MRD) after treatment with dasatinib. The patient expressed the P190BCR-ABL1 isoform and a novel BCR-ABL1 mutation, p.Y440C. The latter is in the C-terminal lobe of the kinase domain, which likely induces deviations in the protein structure and activity and destabilizes its inactive conformation. The treatment was substituted by bosutinib, which binds to the active conformation of the protein, prior to allogeneic bone marrow transplant to overcome the lack of a complete response to dasatinib. These findings strengthen the importance of BCR-ABL1 mutational screening in Ph+ patients, particularly for those who do not achieve complete molecular remission.

Optical and Structural Characterization of a Chronic Myeloid Leukemia DNA Biosensor.

Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.

Multifunctional microfluidic chip for optical nanoprobe based RNA detection - application to Chronic Myeloid Leukemia.

Many diseases have their treatment options narrowed and end up being fatal if detected during later stages. As a consequence, point-of-care devices have an increasing importance for routine screening applications in the health sector due to their portability, fast analyses and decreased cost. For that purpose, a multifunctional chip was developed and tested using gold nanoprobes to perform RNA optical detection inside a microfluidic chip without the need of molecular amplification steps. As a proof-of-concept, this device was used for the rapid detection of chronic myeloid leukemia, a hemato-oncological disease that would benefit from early stage diagnostics and screening tests. The chip passively mixed target RNA from samples, gold nanoprobes and saline solution to infer a result from their final colorimetric properties. An optical fiber network was used to evaluate its transmitted spectra inside the chip. Trials provided accurate output results within 3 min, yielding signal-to-noise ratios up to 9 dB. When compared to actual state-of-art screening techniques of chronic myeloid leukemia, these results were, at microscale, at least 10 times faster than the reported detection methods for chronic myeloid leukemia. Concerning point-of-care applications, this work paves the way for other new and more complex versions of optical based genosensors.

A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification Detection.

Digital microfluidics (DMF) arises as the next step in the fast-evolving field of operation platforms for molecular diagnostics. Moreover, isothermal schemes, such as loop-mediated isothermal amplification (LAMP), allow for further simplification of amplification protocols. Integrating DMF with LAMP will be at the core of a new generation of detection devices for effective molecular diagnostics at point-of-care (POC), providing simple, fast, and automated nucleic acid amplification with exceptional integration capabilities. Here, we demonstrate for the first time the role of coupling DMF and LAMP, in a dedicated device that allows straightforward mixing of LAMP reagents and target DNA, as well as optimum temperature control (reaction droplets undergo a temperature variation of just 0.3 °C, for 65 °C at the bottom plate). This device is produced using low-temperature and low-cost production processes, adaptable to disposable and flexible substrates. DMF-LAMP is performed with enhanced sensitivity without compromising reaction efficacy or losing reliability and efficiency, by LAMP-amplifying 0.5 ng/µL of target DNA in just 45 min. Moreover, on-chip LAMP was performed in 1.5 µL, a considerably lower volume than standard bench-top reactions.

Quantitative real-time monitoring of RCA amplification of cancer biomarkers mediated by a flexible ion sensitive platform.

Ion sensitive field-effect transistors (ISFET) are the basis of radical new sensing approaches. Reliable molecular characterization of specific detection of DNA and/or RNA is vital for disease diagnostics and to follow up alterations in gene expression profiles. Devices and strategies for biomolecular recognition and detection should be developed into reliable and inexpensive platforms. Here, we describe the development of a flexible thin-film sensor for label free gene expression analysis. A charge modulated ISFET based sensor was integrated with real-time DNA/RNA isothermal nucleic acid amplification: Loop-mediated isothermal amplification (LAMP) and Rolling Circle Amplification (RCA) techniques for c-MYC and BCR-ABL1 genes, allowing for the real-time quantification of template. Also, RCA allowed the direct quantification of RNA targets at room temperature, eliminating the requirement for external temperature controllers and overall complexity of the molecular diagnostic approach. This integration between the biological and the sensor/electronic approaches enabled the development of an inexpensive and direct gene expression-profiling platform.

Gold Nanoparticles for Diagnostics: Advances towards Points of Care.

The remarkable physicochemical properties of gold nanoparticles (AuNPs) have prompted developments in the exploration of biomolecular interactions with AuNP-containing systems, in particular for biomedical applications in diagnostics. These systems show great promise in improving sensitivity, ease of operation and portability. Despite this endeavor, most platforms have yet to reach maturity and make their way into clinics or points of care (POC). Here, we present an overview of emerging and available molecular diagnostics using AuNPs for biomedical sensing that are currently being translated to the clinical setting.

Liposomes as Delivery System of a Sn(IV) Complex for Cancer Therapy.

PROPOSE: Tin complexes demonstrate antiproliferative activities in some case higher than cisplatin, with IC50 at the low micromolar range. We have previously showed that the cyclic trinuclear complex of Sn(IV) bearing an aromatic oximehydroxamic acid group [nBu2Sn(L)]3 (L=N,2-dihydroxy-5-[N-hydroxyethanimidoyl]benzamide) (MG85) shows high anti-proliferative activity, induces apoptosis and oxidative stress, and causes destabilization of tubulin microtubules, particularly in colorectal carcinoma cells. Despite the great efficacy towards cancer cells, this complex still shows some cytotoxicity to healthy cells. Targeted delivery of this complex specifically towards cancer cells might foster cancer treatment. METHODS: MG85 complex was encapsulated into liposomal formulation with and without an active targeting moiety and cancer and healthy cells cytotoxicity was evaluated. RESULTS: Encapsulation of MG85 complex in targeting PEGylated liposomes enhanced colorectal carcinoma (HCT116) cancer cell death when compared to free complex, whilst decreasing cytotoxicity in non-tumor cells. Labeling of liposomes with Rhodamine allowed assessing internalization in cells, which showed significant cell uptake after 6 h of incubation. Cetuximab was used as targeting moiety in the PEGylated liposomes that displayed higher internalization rate in HCT116 cells when compared with non-targeted liposomes, which seems to internalize via active binding of Cetuximab to cells. CONCLUSIONS: The proposed formulation open new avenues in the design of innovative transition metal-based vectorization systems that may be further extended to other novel metal complexes towards the improvement of their anti-cancer efficacy, which is usually hampered by solubility issues and/or toxicity to healthy tissues.

Synthesis, characterization, thermal properties and antiproliferative potential of copper(II) 4'-phenyl-terpyridine compounds.

Reactions between 4'-phenyl-terpyridine (L) and several Cu(II) salts (p-toluenesulfonate, benzoate and o-, m- or p-hydroxybenzoate) led to the formation of [Cu(p-SO3C6H4CH3)L(H2O)2](p-SO3C6H4CH3) (1), [Cu(OCOPh)2L] (2), [Cu(o-OCOC6H4OH)2L] (3), [Cu(m-OCOC6H4OH)2L]4·MeOH (·MeOH) and [Cu(p-OCOC6H4OH)2L]5·2H2O (·2H2O), which were characterized by elemental and TG-DTA analyses, ESI-MS, IR spectroscopy and single crystal X-ray diffraction, as well as by conductivimetry. In all structures the Cu atoms present N3O3 octahedral coordination geometries, which, in 2-5, are highly distorted as a result of the chelating-bidentate mode of one of the carboxylate ligands. Intermolecular π···π stacking interactions could also be found in 2-5 (in the 3.569-3.651 Å range and involving solely the pyridyl rings). Medium-strong hydrogen bond interactions lead to infinite 1D chains (in 1 and 4) and to an infinite 2D network (in 5). Compounds 1 and 4 show high in vitro cytotoxicity towards HCT116 colorectal carcinoma and HepG2 hepatocellular carcinoma cell lines. The antiproliferative potential of compound 1 is due to an increase of the apoptotic process that was confirmed by Hoechst staining, flow cytometry and RT-qPCR. All compounds able to non-covalently intercalate the DNA helix and induce in vitro pDNA double-strand breaks in the absence of H2O2. Concerning compound 1, the hydroxyl radical and singlet oxygen do not appear to be involved in the pDNA cleavage process and the fact that this cleavage also occurs in the absence of molecular oxygen points to a hydrolytic mechanism of cleavage.